Tumor antigen

ABSTRACT

A gene, which codes for a tumor antigen that is recognized by a cytotoxic T-lymphocyte (CTL) and/or induces a CTL in an HLA-A2-restricted manner, was isolated from a cDNA library of the human glioma cell strain KNS60 and identified. In addition, a peptide having the epitope of the tumor antigen was found, based on the tumor antigen coded by the gene obtained.

FIELD OF THE INVENTION

The present invention relates to a tumor antigen, more specifically, itrelates to a peptide or a polypeptide that is recognized bytumor-specific cytotoxic T-lymphocytes; a polynucleotide that codes forthe peptide or the polypeptide, or a polynucleotide that is thecomplementary strand thereof; a recombinant vector that comprises thepolynucleotide; a transformant that comprises the recombinant vector; anantibody that is directed against the peptide or the polypeptide; acompound that interacts with the peptide, the polypeptide, or thepolynucleotide; a medicament containing the peptide and/or thepolypeptide; a preventive agent and/or a therapeutic agent for multiplesclerosis containing the peptide and/or the polypeptide; a cancervaccine or a cytotoxic T-lymphocyte inducing agent that contains thepeptide and/or the polypeptide; a pharmaceutical composition thatcontains one or more species thereof; a method for preparing the peptideor the polypeptide; a method for identifying the compound that interactswith the peptide, the polypeptide, or the polynucleotide; a method forinducing cytotoxic T-lymphocytes by using the peptide or thepolypeptide; a method for measuring the peptide, the polypeptide, or thepolynucleotide; as well as a reagent kit for use in the identificationmethod or the measurement method.

BACKGROUND OF THE INVENTION

In eliminating cancer in vivo, the immune system, and in particular,cytotoxic T-lymphocytes involved in cell-mediated immunity, play animportant role. For instance, infiltration of a cytotoxic T-lymphocytethat demonstrates cytotoxicity against tumor cells has been observed ata tumor site in cancer patients (Non-Patent Reference 1). The targetmolecule of such tumor-specific cytotoxic T-lymphocytes, or so-calledtumor antigens, was discovered for the first time in melanoma. The tumorantigen that is generated in a tumor cell is degraded in the cell into apeptide that consists of 8 to 11 amino acids, or a so-called tumorantigen peptide, which binds to a molecule of human leukocyte antigen(hereinafter abbreviated as HLA) which is a major histocompatibilitycomplex (hereinafter abbreviated as MHC), and is presented on thesurface of the tumor cell. The cytotoxic T-lymphocyte recognizes thecomplex comprising this HLA molecule and the tumor antigen peptide,releases a soluble factor, for example, a cytokine, such asinterferon-γ, and then lyses the tumor cell. That is to say, thecytotoxic T-lymphocyte demonstrates a cytotoxic activity by recognizingtumor cells in an HLA-restricted manner.

HLA is a cell membrane antigen that is expressed in almost alleukaryotic cells. HLA is broadly divided into class I antigens and classII antigens; HLA recognized together with the antigen peptide bycytotoxic T-lymphocytes is a class I antigen. HLA class I antigens arefurther classified into HLA-A, HLA-B, HLA-C, and the like; and inhumans, different eukaryotic cells have different quantities of HLA-A,HLA-B, and HLA-C. In addition, a genetic polymorphism therefor has beenreported. For instance, for HLA-A there are such polymorphisms as A1,A2, A24, and A26; for HLA-B there are such polymorphisms as B8, B27, andB46; and for HLA-C there are such polymorphisms as Cw3 and Cw6. Thus,the types of HLA that each individual possesses are not necessarilyidentical. The HLA-A2 allele, which is one of polymorphism of the HLA-Asub-region, is found in approximately 40% of Japanese, approximately 53%of Chinese, approximately 49% of North American Caucasians,approximately 38% of South American Caucasians, and approximately 23% ofAfrican Blacks.

When the cytotoxic T-lymphocyte recognizes a complex of an HLA class Iantigen and a tumor antigen peptide, it also recognizes the HLA typethereof. In addition, a motif (regular arrangement), which is differentdepending on the HLA type, is known to exist in the amino acid sequenceof the tumor antigen peptide that binds to the HLA molecule. Since apeptide that binds to the HLA molecule differs depending on the type ofHLA, it is necessary to select a peptide that binds to HLA of each typein order to induce and/or activate an antigen-specific cytotoxicT-lymphocyte using a tumor antigen peptide.

In recent years, molecules that are involved in specific immunity, suchas tumor rejection antigen genes and T-cell antigen receptors (T-cellreceptors), have been identified in a variety of cancers, for instance,in melanoma, esophageal cancer, and the like, and specificimmunotherapies using peptides are being examined in advanced cancersand metastatic cancers (Non-Patent References 2, 3, 4, 5, 6, and 7).

Currently, in Europe and America, cancer vaccine therapies are beingdeveloped, wherein cytotoxic T-lymphocytes inside the body of a cancerpatient are activated by administration of a tumor antigen, and resultsfrom clinical studies have been reported for melanoma specific tumorantigens. For example, as a result of subcutaneously administratingmelanoma antigen gp100 peptide to melanoma patients and intravascularlyadministrating interleukin-2, reduction of tumor was observed in 42% ofthe patients (Non-Patent Reference 8). Thus, an effective cancer therapyeffect can be expected from a tumor antigen, when it is used as a cancervaccine.

However, when the diversity of cancers is considered, it is impossibleto treat all cancers using a cancer vaccine consisting of only one typeof tumor antigen. Diversity of the type or the tissue of cancer cellsgives diversity of the type or the amount of a tumor antigen beingexpressed in the cancer cells. Actually, it has been reported thatimmunotherapy, using a plurality of peptides (multi-peptide basedimmunotherapy), is effective in cancer therapy (Non-Patent Reference 9,10, and 11).

In addition, given that the type of tumor antigen peptide that functionsin each individual differs due to the polymorphism of the HLA gene, itis important to identify a tumor antigen peptide that induces and/oractivates an antigen-specific cytotoxic T-lymphocyte restricted for eachHLA type in order to obtain a high effectiveness in cancer therapy.

Obviously, a cancer vaccine therapy that activates cytotoxicT-lymphocytes using a single tumor antigen will bring some therapeuticeffects for a cancer having this tumor antigen. However, in cancertherapy, in order to induce and/or activate antigen-specific cytotoxicT-lymphocytes, and also to obtain high therapeutic effectivenesscorresponding to the diversity of cancers, it is important to discoverand use numerous novel tumor antigens that correspond to theHLA-restriction and diversity of cancers.

The references cited in the description of this background art arelisted below.

-   Non-Patent Reference 1: Archives of Surgery, 1990, Volume 126, pp.    200-205.-   Non-Patent Reference 2: Science, 1991, Volume 254, pp. 1643-1647.-   Non-Patent Reference 3: Journal of Experimental Medicine, 1996,    Volume 183, pp. 1185-1192.-   Non-Patent Reference 4: Journal of Immunology, 1999, Volume 163, pp.    4994-5004.-   Non-Patent Reference 5: Proceedings of the National Academy of    Sciences of the United States of America, 1995, Volume 92, pp.    432-436.-   Non-Patent Reference 6: Science, 1995, Volume 269, pp. 1281-1284.-   Non-Patent Reference 7: Journal of Experimental Medicine, 1997,    Volume 186, pp. 785-793.-   Non-Patent Reference 8: Nature Medicine, 1998, Volume 4, pp.    321-327.-   Non-Patent Reference 9: Clinical Cancer Research, 2001, Volume 7,    pp. 3950-3962.-   Non-Patent Reference 10: Journal of Clinical Oncology, 2001, Volume    19, pp. 3836-3847.-   Non-Patent Reference 11: Nature Medicine, 1998, Volume 4, pp.    328-332.

SUMMARY OF THE INVENTION

One aspect of the present invention is a peptide having the amino acidsequence set forth in any one of SEQ ID NO: 1 to 13 in the SequenceListing.

One additional aspect of the present invention is a polypeptide havingthe amino acid sequence set forth in any one of SEQ ID NO: 14 to 18 inthe Sequence Listing.

One further aspect of the present invention is a peptide having theamino acid sequence set forth in any one of SEQ ID NO: 1 to 13 in theSequence Listing, wherein the peptide is recognized by a cytotoxicT-lymphocyte and/or induces a cytotoxic T-lymphocyte.

A still further aspect of the present invention is the aforementionedpeptide, wherein being recognized by a cytotoxic T-lymphocyte and/orinducing a cytotoxic T-lymphocyte is being recognized by a cytotoxicT-lymphocyte in an HLA-A2-restricted manner and/or inducing a cytotoxicT-lymphocyte in an HLA-A2-restricted manner.

One additional and further aspect of the present invention is apolypeptide having the amino acid sequence set forth in any one of SEQID NO: 14 to 18 in the Sequence Listing, wherein the polypeptide isrecognized by a cytotoxic T-lymphocyte and/or induces a cytotoxicT-lymphocyte.

One aspect of the present invention, in addition, is the aforementionedpolypeptide, wherein being recognized by a cytotoxic T-lymphocyte and/orinducing a cytotoxic T-lymphocyte is being recognized by a cytotoxicT-lymphocyte in an HLA-A2-restricted manner and/or inducing a cytotoxicT-lymphocyte in an HLA-A2-restricted manner.

One aspect of the present invention is, furthermore, a medicamentcomprising one or more peptides selected from peptides having therespective amino acid sequences set forth in SEQ ID NO: 1 to 13 in theSequence Listing, and/or one or more polypeptides selected frompolypeptides having the respective amino acid sequences set forth in SEQID NO: 14 to 18 in the Sequence Listing.

One still further aspect of the present invention is a cancer vaccinethat contains one or more peptides selected from peptides having therespective amino acid sequences set forth in SEQ ID NO: 1 to 13 in theSequence Listing, and/or one or more polypeptides selected frompolypeptides having the respective amino acid sequences set forth in SEQID NO: 14 to 18 in the Sequence Listing.

One aspect of the present invention is, furthermore, the aforementionedcancer vaccine, which is used in the treatment of a brain tumor.

In addition, one aspect of the present invention is an agent forinducing a cytotoxic T-lymphocyte that contains one or more peptidesselected from peptides having the respective amino acid sequences setforth in SEQ ID NO: 1 to 13 in the Sequence Listing, and/or one or morepolypeptides selected from polypeptides having the respective amino acidsequences set forth in SEQ ID NO: 14 to 18 in the Sequence Listing.

Furthermore, one aspect of the present invention is a method forinducing a cytotoxic T-lymphocyte comprising using the use of one ormore peptides selected from peptides having the respective amino acidsequences set forth in SEQ ID NO: 1 to 13 in the Sequence Listing,and/or one or more polypeptides selected from polypeptides having therespective amino acid sequences set forth in SEQ ID NO: 14 to 18 in theSequence Listing. Furthermore, one aspect of the present invention is amethod for inducing a cytotoxic T-lymphocyte comprising the steps of:

-   -   i) incubating an antigen-presenting cell that retains HLA-A2        with a peptide having the amino acid sequence set forth in any        one of SEQ ID NO: 1 to 13 in the Sequence Listing; or    -   ii) expressing a polypeptide having the amino acid sequence set        forth in any one of SEQ ID NO: 14 to 18 in the Sequence Listing        in an antigen-presenting cell that retains HLA-A2; and    -   iii) using the cell obtained in the aforementioned step i) or        the aforementioned step ii) for stimulating a group of cells        that contain a precursor cell of the cytotoxic T-lymphocyte.

Furthermore, one aspect of the present invention is the aforementionedmedicament, which is used in the prevention and/or the treatment ofmultiple sclerosis.

One aspect of the present invention is, in addition, a polynucleotidehaving a nucleotide sequence that codes for a peptide having the aminoacid sequence set forth in any one of SEQ ID NO: 1 to 13 in the SequenceListing, or a polypeptide having the amino acid sequence set forth inany one of SEQ ID NO: 14 to 18 in the Sequence Listing, or acomplementary nucleotide sequence thereof.

One aspect of the present invention is, furthermore, a polynucleotidehaving a nucleotide sequence set forth in any of SEQ ID NO: 19 to 23 inthe Sequence Listing, or a complementary nucleotide sequence thereof.

One still further aspect of the present invention is a polynucleotidehaving the nucleotide sequence set forth in any one of SEQ ID NO: 19 to23 in the Sequence Listing, wherein the nucleotide sequence is such thatthe polypeptide coded by said nucleotide sequence is recognized by acytotoxic T-lymphocyte, and/or induces a cytotoxic T-lymphocyte, or acomplementary nucleotide sequence thereof.

One aspect of the present invention is, in addition, the aforementionedpolynucleotide wherein being recognized by a cytotoxic T-lymphocyteand/or inducing a cytotoxic T-lymphocyte is being recognized by acytotoxic T-lymphocyte in an HLA-A2-restricted manner and/or inducing acytotoxic T cell in an HLA-A2-restricted manner.

In addition, one aspect of the present invention is a polynucleotidethat hybridizes under stringent conditions with any of theaforementioned polynucleotides.

Furthermore, one aspect of the present invention is a recombinant vectorthat comprises any of the aforementioned polynucleotides.

Moreover, one aspect of the present invention is the aforementionedrecombinant vector, wherein the recombinant vector is a recombinantexpression vector.

In addition, one aspect of the present invention is a transformant thathas been transformed by any of the aforementioned recombinant vectors.

One aspect of the present invention is, in addition, a method forpreparing any of the aforementioned peptides or any of theaforementioned polypeptides, wherein the method comprises a step ofculturing a transformant that has been transformed by a recombinantexpression vector containing any of the aforementioned polynucleotides.

One aspect of the present invention is, furthermore, an antibody thatimmunologically recognizes any of the aforementioned peptides and/or anyof the aforementioned polypeptides.

Moreover, one aspect of the present invention is a method foridentifying: a compound that interacts with the aforementioned peptideor the aforementioned polypeptide and/or an HLA-A2 molecule, and atleast enhances the recognition of the peptide or the polypeptide by anHLA-A2-restricted cytotoxic T-lymphocyte; and/or a compound thatinteracts with any of the aforementioned polynucleotides and enhancesthe expression thereof, wherein the method comprises using at least oneof: the aforementioned peptides; the aforementioned polypeptides; any ofthe aforementioned polynucleotides; any of the aforementionedrecombinant vectors; the aforementioned transformants; or theaforementioned antibodies.

In addition, one aspect of the present invention is a compound that isidentified by the method described above.

One aspect of the present invention is, in addition, a compound thatenhances the recognition of at least one of the aforementioned peptidesor the aforementioned polypeptides by an HLA-A2-restricted cytotoxicT-lymphocyte.

One aspect of the present invention is, furthermore, a compound thatinteracts with any of the aforementioned polynucleotides and enhancesthe expression thereof.

One still further aspect of the present invention is a pharmaceuticalcomposition for use in cancer therapy that contains an effective dose ofat least one of: any of the aforementioned peptides; any of theaforementioned polypeptides; any of the aforementioned polynucleotides;any of the aforementioned recombinant vectors; the aforementionedtransformants; the aforementioned antibodies; or any of theaforementioned compounds.

One aspect of the present invention is, furthermore, a pharmaceuticalcomposition for use in the prevention and/or treatment of multiplesclerosis that contains an effective dose of at least one of: any of theaforementioned peptides; any of the aforementioned polypeptides; any ofthe aforementioned polynucleotides; any of the aforementionedrecombinant vectors; the aforementioned transformants; theaforementioned antibodies; or any of the aforementioned compounds.

In addition, one aspect of the present invention is a method forquantitatively and/or qualitatively measuring: any of the aforementionedpeptides; any of the aforementioned polypeptides; or any of theaforementioned polynucleotides.

Furthermore, one aspect of the present invention is the use of theaforementioned measurement method, which is used in cancer screening.

A still further aspect of the present invention is a reagent kitcomprising at least one of: any of the aforementioned peptides; any ofthe aforementioned polypeptides; any of the aforementionedpolynucleotides; or the aforementioned antibodies.

In addition, one aspect of the present invention is a reagent kit foruse in any of the aforementioned methods, wherein the reagent kitcomprises at least one of the following: any of the aforementionedpeptides; any of the aforementioned polypeptides; any of theaforementioned polynucleotides; or the aforementioned antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

In the figures, * indicates that a significant difference (P<0.05) wasobserved in the two-tailed Student t-test.

FIG. 1 shows that the cDNA clone 8B6, derived from human glioma cellKNS60, was recognized in a plasmid dose-dependent manner and also in anHLA-A2-restricted manner by a cytotoxic T-lymphocyte strain, OK-CTL, andthat it enhanced the production of interferon-γ from the OK-CTL.

FIG. 2 shows that the cDNA clone 2G2, derived from the human glioma cellKNS60, was recognized in a plasmid dose-dependent manner and also in anHLA-A2-restricted manner by the cytotoxic T-lymphocyte strain OK-CTL,and that it enhanced the production of interferon-γ from the OK-CTL.

FIGS. 3A, 3B, and 3C show, respectively, that P101 (SEQ ID NO: 1), P102(SEQ ID NO: 2), and P103 (SEQ ID NO: 3) which are peptides derived fromclone 8B6, were recognized by the HLA-A2-restricted cytotoxicT-lymphocyte strain OK-CTL in a peptide dose-dependent manner, and thatthey enhanced the production of interferon-γ from the OK-CTL.

FIGS. 4A, 4B, and 4C show, respectively, that P104 (SEQ ID NO: 4 in theSequence Listing), P105 (SEQ ID NO: 5 in the Sequence Listing), and P106(SEQ ID NO: 6 in the Sequence Listing) which are peptides derived fromclone 2G2, were recognized by the HLA-A2-restricted cytotoxicT-lymphocyte strain OK-CTL in a peptide dose-dependent manner, and thatthey enhanced the production of interferon-γ from the OK-CTL.

FIG. 5 shows that each of seven peptides (P1, P2, P3, P6, P14, P18, andP19), derived from clone 4G3, clone 7H9, or clone 1B10, were recognizedby the HLA-A2-restricted cytotoxic T-lymphocyte strain OK-CTL in apeptide dose-dependent manner, and that they enhanced the production ofinterferon-γ from the OK-CTL.

FIGS. 6A and 6B show that, when peripheral blood mononuclear cells(PBMC) derived from a metastatic brain tumor patient and PBMC derivedfrom a meningioma patient, respectively, were stimulated with P101 (SEQID NO: 1), P102 (SEQ ID NO: 2), and P103 (SEQ ID NO: 3), which arederived from clone 8B6, or P104 (SEQ ID NO: 4 in the Sequence Listing),P105 (SEQ ID NO: 5 in the Sequence Listing), and P106 (SEQ ID NO: 6 inthe Sequence Listing), which are peptides derived from clone 2G2, theyrecognized T2 cells (HLA-A2⁺) that had been pulsed with the eachcorresponding peptide, and as a result, production of interferon-γ fromPBMC was enhanced.

FIGS. 7A, 7B, 7C, and 7D show that peripheral blood mononuclear cells,derived from a metastatic brain tumor patient, which were stimulatedwith peptide P101 (SEQ ID NO: 1 in the Sequence Listing), which isderived from clone 8B6, peptide P103 (SEQ ID NO: 3 in the SequenceListing), which is derived from clone 8B6, peptide P106 (SEQ ID NO: 6 inthe Sequence Listing), which is derived from clone 2G2, and peptide P104(SEQ ID NO: 4 in the Sequence Listing), which is derived from clone 2G2,respectively recognized HLA-A2+tumor cell strain KNS60, indicatedcytotoxic activity, and lysed the tumor cell strain, but they did notrecognize HLA-A2⁻ tumor cell strain KALS-1 or any normal cells(PHA-blast and EB-BC).

FIGS. 8A and 8B show that peripheral blood mononuclear cells (PBMC),derived from different brain tumor patients, which were stimulated withP101 (SEQ ID NO: 1), P102 (SEQ ID NO: 2), or P103 (SEQ ID NO: 3), whichare peptides derived from clone 8B6, recognized T2 cells (HLA-A2⁺) thathad been pulsed with each corresponding peptide and/or tumor cell strain(HLA-A2⁺), and as a result, production of interferon-γ from the PBMC wasenhanced. In these figures, HIV, EB, and flu indicate control peptidesderived from an immunodeficiency virus, an Epstein-Barr virus, and aninfluenza virus, respectively.

FIG. 9 shows that peripheral blood mononuclear cells (PBMC), derivedfrom a brain tumor patient, when stimulated with P104 (SEQ ID NO: 4 inthe Sequence Listing), P105 (SEQ ID NO: 5 in the Sequence Listing), andP106 (SEQ ID NO: 6 in the Sequence Listing), which are peptides derivedfrom clone 2G2, recognized T2 cells (HLA-A2⁺) that had been pulsed witheach corresponding peptide and/or tumor cell strain (HLA-A2⁺), and as aresult, production of interferon-γ from the PBMC was enhanced. In thisfigure, HIV, EB, and flu indicate control peptides derived from animmunodeficiency virus, an Epstein-Barr virus, and an influenza virus,respectively.

FIGS. 10A and 10B show that peripheral blood mononuclear cells (PBMC),derived from different multiple sclerosis patients, when stimulated withP101 (SEQ ID NO: 1), P102 (SEQ ID NO: 2), or P103 (SEQ ID NO: 3), whichare peptides derived from clone 8B6, recognized T2 cells that had beenpulsed with each corresponding peptide, and as a result, production ofinterferon-γ from the PBMC was enhanced, but the PBMC did not recognizethe tumor cells. In these figures, HIV, EB, and flu indicate controlpeptides derived from an immunodeficiency virus, an Epstein-Barr virus,and an influenza virus, respectively.

FIG. 11 shows that peripheral blood mononuclear cells (PBMC), derivedfrom a multiple sclerosis patient, when stimulated with P104 (SEQ ID NO:4 in the Sequence Listing), P105 (SEQ ID NO: 5 in the Sequence Listing),and P106 (SEQ ID NO: 6 in the Sequence Listing), which are peptidesderived from clone 2G2, recognized T2 cells that had been pulsed witheach corresponding peptide, and as a result, production of interferon-γfrom the PBMC was enhanced, but the PBMC did not recognize the tumorcells. In this figure, HIV, EB, and flu indicate control peptidesderived from an immunodeficiency virus, an Epstein-Barr virus, and aninfluenza virus, respectively.

FIGS. 12A and 12B show that production of interferon-γ from peripheralblood mononuclear cells, derived from a healthy subject, was inhibitedby anti-HLA-class II antibody, anti-CD4 antibody, or anti-HLA-A2antibody; the peripheral blood mononuclear cells having been stimulatedwith peptide P104 (SEQ ID NO: 4 in the Sequence Listing) and peptideP106 (SEQ ID NO: 6 in the Sequence Listing), respectively, which arepeptides derived from clone 2G2; and the production of interferon-γhaving been enhanced by the peripheral blood mononuclear cellsrecognizing T2 cells that have been pulsed with the correspondingpeptide.

FIGS. 13A and 13B show that production of interferon-γ from CD4-positivecells, purified from peripheral blood mononuclear cells derived from ahealthy subject, was inhibited by an anti-HLA-class I antibody or ananti-HLA-class II antibody; the peripheral blood mononuclear cellshaving been stimulated with peptide P104 (SEQ ID NO: 4 in the SequenceListing) and peptide P106 (SEQ ID NO: 6 in the Sequence Listing),respectively, which are peptides derived from clone 2G2; and theproduction of interferon-γ having been enhanced by CD4-positive cellsrecognizing T2 cells that had been pulsed with each correspondingpeptide respectively.

FIGS. 14A and 14B show, respectively, the results obtained byquantifying specific IgE antibodies and specific IgG antibodies againstP102 (SEQ ID NO: 2) or P103 (SEQ ID NO: 3), which are peptides derivedfrom clone 8B6, or P104 (SEQ ID NO: 4) or P106 (SEQ ID NO: 6), which arepeptides derived from clone 2G2, in the serum of a multiple sclerosispatient.

FIGS. 15A and 15B show, respectively, the results obtained byquantifying specific IgE antibodies and specific IgG antibodies againstP102 (SEQ ID NO: 2) or P103 (SEQ ID NO: 3), which are peptides derivedfrom clone 8B6, or P104 (SEQ ID NO: 4) or P106 (SEQ ID NO: 6), which arepeptides derived from clone 2G2, in the serum of a brain tumor patient.

FIGS. 16A and 16 show, respectively, the results obtained by quantifyingspecific IgE antibodies and specific IgG antibodies against P102 (SEQ IDNO: 2) or P103 (SEQ ID NO: 3), which are peptides derived from clone8B6, or P104 (SEQ ID NO: 4) or P106 (SEQ ID NO: 6), which are peptidesderived from clone 2G2, in the serum of a healthy subject.

DETAILED DESCRIPTION OF THE INVENTION

The present invention claims priority from Japanese Patent ApplicationNo. 2001-333219 which is incorporated herein by reference.

In order to understand the present invention, the terminology used inthe present specification will first be explained. A tumor antigen meansa protein or peptide that a tumor cell possesses, which may berecognized by a tumor-specific cytotoxic T-lymphocyte and/or may inducea cytotoxic T-lymphocyte. In addition, a tumor antigen peptide means apeptide generated by degrading of the tumor antigen inside a tumor cell,and this peptide may be recognized by a tumor-specific cytotoxicT-lymphocyte and/or induce a cytotoxic T-lymphocyte by binding to an HLAmolecule and being presented on the surface of the cell. Furthermore,the site of an amino acid sequence that a tumor antigen possesses, whichmay induce and/or activate a tumor-specific cytotoxic T-lymphocyte, iscalled a tumor antigen epitope (tumor antigen determinant).

Here, to “recognize” means that a recognizing entity discerns ato-be-recognized target from others and may bind to the recognizedtarget. In particular, in the present specification, recognition of atumor cell or a tumor antigen peptide by a cytotoxic T-lymphocyte meansthat a cytotoxic T-lymphocyte binds to a tumor antigen peptide presentedby an HLA molecule via a T-cell antigen receptor (hereinafter alsoabbreviated as TCR). To “activate” means to further enhance or bringinto action an entity or a state that has a given activity or effect. Inparticular, in the present specification, activation of a cytotoxicT-lymphocyte means that a cytotoxic T-lymphocyte generates, for example,IFN-γ as a result of recognizing an antigen presented by an HLAmolecule, or that a cytotoxic T-lymphocyte demonstrates a cytotoxicactivity against a target cell (also called target) that has beenrecognized. To “induce” means to generate a given activity or effectfrom an entity or a state that substantially lacks the activity or theeffect. In particular, in the present specification, to induce anantigen-specific cytotoxic T-lymphocyte means to cause a cytotoxicT-lymphocyte that specifically recognizes a given antigen todifferentiate and/or to proliferate in vitro or in vivo. In addition, inthe present specification, a “cytotoxic T-lymphocyte inducing agent”means an agent that demonstrates an effect, wherein a state where CD8positive T-lymphocytes that specifically recognize a given antigen isnot present or is present only at an extremely low ratio, is changed toa state where cytotoxic T-lymphocytes that recognize the antigen arepresent at an extremely high ratio.

In the present specification, a long chain peptide among any of peptidescontaining two or more amino acids bound to one another through apeptide bond or a modified peptide bond, is called a polypeptide. Forinstance, in the present specification, proteins are also included aspolypeptides. In addition, short chain peptides, also referred to asoligopeptides and oligomers, are simply called peptides. In thefollowing, when an amino acid sequence is represented, it may berepresented by one letter, or it may be represented by three letters.

Other technical and scientific terms used herein have the meaningscommonly understood by one of ordinary skill in the art to which thepresent invention pertains, unless otherwise defined. Reference is madeherein to various methodologies known to those of ordinary skill in theart. Publications and other materials setting forth such knownmethodologies to which reference is made are incorporated herein byreference in their entireties.

Hereinafter, the present invention will be described in more detail forthe various embodiments of the present invention.

The following detailed description is illustrative, and its purpose ismerely explanatory and does not limit the present invention in any way.

The peptide provided in the present invention is a partial peptide thatis contained in a polypeptide coded by a cDNA that has been isolated andidentified from a cDNA library of KNS60 cells (Human Cell, 1990, Volume3, pp. 255-256), which is a human brain tumor cell strain, using thegene expression cloning method. When the cDNA is introduced into a celland expressed, the cell is recognized by a tumor-specific cytotoxicT-lymphocyte, and can induce and/or activate the cytotoxic T-lymphocyte.More concretely, when expressed in a cell that possesses HLA-A2, it isrecognized by the tumor-specific cytotoxic T-lymphocyte in anHLA-A2-restricted manner, and can induce and/or activate the cytotoxicT-lymphocyte.

The aforementioned peptide that is contained in the polypeptide coded bysuch cDNA has a characteristic that it is recognized by a tumor-specificcytotoxic T-lymphocyte and can induce and/or activate the cytotoxicT-lymphocyte. More concretely, it is recognized by the tumor-specificcytotoxic T-lymphocyte in an HLA-A2-restricted manner, and can induceand/or activate the cytotoxic T-lymphocyte.

Isolation and identification of the above-mentioned cDNA were carriedout as follows. First, an HLA-A2-restricted and tumor-specific cytotoxicT-lymphocyte, which can be activated by recognizing the HLA-A2 and atumor antigen peptide, was established from tumor-infiltratinglymphocytes (hereinafter abbreviated as TIL) of a colon cancer patient(HLA-A0207/3101) by methods set forth in existing reports (Non-PatentReference 4). Hereinafter, this cell is referred to as OK-CTL. The cellsurface markers of OK-CTL are CD3⁺CD4⁻CD8⁺, and antigen recognitionthereof is HLA-A gene locus restricted. That is to say, it recognizes anHLA-A2-positive (hereinafter noted as HLA-A2⁺) cell, but does notrecognize an HLA-A2-negative (hereinafter noted as HLA-A2⁻) cell.HLA-A2-positive means that the antigen specificity of the HLA-A alleleis A2. HLA-A2-negative means that the antigen specificity of the HLA-Aallele is other than A2.

Next, COS-7 cells were cotransfected with the cDNA derived from KNS60tumor cells and HLA-A0207 cDNA, and among the cells wherein thetransgenes were expressed, those that enhance the production ofinterferon-γ (hereinafter may be abbreviated as IFN-γ) from OK-CTL wereselected. As a result, five types of cDNA clones (from SEQ ID NO: 19 to23) that code for gene products that are recognized by the OK-CTL in anHLA-A2-restrictive manner were obtained.

When a homology search was carried out for these nucleotide sequencesagainst existing databases, such as GenBank, human genes that are highlyhomologous to clone 8B6 (SEQ ID NO: 19) and clone 2G2 (SEQ ID NO: 20)were found; however, regarding three types of genes in total, such asclone 4G3 (SEQ ID NO: 21), clone 7H9 (SEQ ID NO: 22), and clone 1B10(SEQ ID NO: 23), highly homologous genes were not found (see Table 1).Although the nucleotide sequences of the human genes highly homologousto clone 8B6 (SEQ ID NO: 19) or clone 2G2 (SEQ ID NO: 20) and thededuced amino acid sequences thereof are disclosed, there is no reportthat they code for tumor antigens, nor are these disclosed in the opendatabase of the National Center for Biotechnology Information (NCBI)when it was searched as of 7 Nov., 2001.

The nucleotide sequences of clone 8B6 (SEQ ID NO: 19) and clone 2G2 (SEQID NO: 20) and the nucleotide sequence of clone 4G3 (SEQ ID NO: 21),clone 7H9 (SEQ ID NO: 22), and clone 1B10 (SEQ ID NO: 23), wereregistered with the DNA Data Bank of Japan (DDBJ) of the NationalInstitute of Genetics on 24 Apr., 2001, and on 6 Jul., 2001,respectively; however, they were not disclosed as of 30 Oct., 2001.

The genes obtained in the present invention are genes that code fortumor antigens that are recognized by HLA-A2-restricted cytotoxicT-lymphocytes (hereinafter may be abbreviated as CTL). These genes codefor the amino acid sequences set forth in SEQ ID NO: 14 to 18,respectively, in the Sequence Listing (see Table 1). These genes, whenexpressed in cells as mentioned above, are recognized by anHLA-A2-restricted CTL and can activate the CTL. TABLE 1 Clone Polypep-(base pairs; tide coded bp) SEQ by gene SEQ SEQ Amino acid Highlyhomologous [Accession ID (amino ID Active ID sequence of the genenumber] NO acids) NO peptide NO peptide [Accession number] 8B6 19 PP 8B614 P 101 1 PIMAFRWVT 1,3-N- (2292) (204) P 102 2 IMSRDLVPRIacetylgalactosam- [AB060691] P 103 3 NLLKVNIHI inyl transferase (GALT3)[AF154848] 2G2 20 PP 2G2 15 P 104 4 FLPHHFQALHV human ADP- (1392) (201)P 105 5 ALHVVVIGL ribosylation [AB060692] P 106 6 GITFQVWDV factor 4L(ARF4) [NM001661] 4G3 21 PP 4G3 16 P 1 7 CLGEEVLET — (701) (73) P 2 8IIGFFCYT [AB065085] P 3 9 GIHLACFVEV 7H9 22 PP 7H9 17 P 6 10 ILWKEKNSA —(1848) (111) [AB065086] 1B10 23 PP 1B10 18 P 14 11 NLVSLFSRYV — (2039)(111) P 18 12 NQWTEVMFMA [AB065087] P 19 13 VMFMATRELL

A peptide having the amino acid sequence listed in any one of SEQ ID NO:1 to 13 in the Sequence Listing was obtained by selecting a peptide thatwas recognized by the CTL in an HLA-A2-restricted manner from peptidesthat were designed and synthesized, as peptides conformable toHLA-A2-binding motifs, based on amino acid sequences that are coded bythe aforementioned genes or homologous genes thereof. It is known that atumor antigen peptide that is capable of binding to an HLA-A2 moleculehas a motif (regular arrangement) in its amino acid sequence. Thus, anHLA-A2-binding motif was first retrieved from the Internet site<http://bimas.dcrt.nih.gov//molbio/hla_bind/>, and the amino acidsequence that conforms to this motif was specified in the amino acidsequences that are coded by the aforementioned genes, as well as in theamino acid sequence of the gene products from the genes that are highlyhomologous to the aforementioned genes. Based on this result, different9- or 10-mer peptides that have the HLA-A2-binding motif were designedand synthesized. T2 cells (HLA-A2⁺), having been pulsed with each of thesynthesized peptides, and OK-CTL were co-cultured, and IFN-γ producedfrom this OK-CTL was measured. Among the synthesized peptides, 13 typesof peptides (SEQ ID NO: 1 to 13) were recognized by the OK-CTL in adose-dependent manner, and they enhanced production of IFN-γ from theOK-CTL. In addition, these peptides induced CTL from the peripheralblood mononuclear cells (hereinafter may be abbreviated as PBMCs) thatwere obtained from a cancer patient. The induced CTL recognized thetarget cells, that is, T2 cells (HLA-A2⁺) that had been pulsed with thecorresponding peptide, and/or HLA-A2⁺ tumor cells produced IFN-γ, andlysed the target cells. Thus, 13 types of tumor antigen peptides thatcan induce and/or activate CTL were obtained in the present invention.

Furthermore, among these thirteen types of peptides, when each of thepeptides listed as SEQ ID NO: 1 to 6 in the Sequence Listing wereco-cultured with the PBMCs obtained from a multiple sclerosis(hereinafter may be abbreviated as MS) patient or a healthy subject, thePBMCs recognized T2 cells (HLA-A2⁺) which were pulsed with thecorresponding peptide, and enhanced the production of IFN-γ, but did notrecognize HLA-A2⁺tumor cells. Since recognition of T2 cells, which werepulsed with the peptide, by the stimulated PBMCs was inhibited byanti-CD4 antibodies, and CD4⁺cells that were purified from thestimulated PBMCs using anti-CD4 antibodies recognized each correspondingpeptide, the cells that may recognize each peptide induced by thestimulation of the aforementioned peptide in the peripheral bloodmononuclear cells from a healthy subject or a multiple sclerosis patientare thought to be, not CD8⁺ CTL, but CD4⁺ T-lymphocytes. Generally, CD4⁺T-lymphocytes are known to recognize complexes of MHC class II moleculesand peptides on antigen-presenting cells and produce various kinds ofcytokines, for instance, IFN-γ. However, recently, a case in which aCD4⁺ cell recognizes an antigen in a class I-restricted manner has beenreported (Cancer Research, 1999, Volume 59, pp. 6230-6238), and thusthere is a possibility that the induced CD4⁺ cells described aboverecognize antigens in a class I-restricted manner.

Multiple sclerosis is a representative demyelination disease of thecentral nervous system; with the pathology and the course thereof beingclinically and pathologically complex, and demonstrating diversity(Martin, R. et al., Nature Immunology, 2001, Volume 2, pp. 785-788). Inaddition, the onset mechanism thereof is complex and has not yet beenelucidated; however, it is said to be an autoimmune disease in whichencephalitogenic T-lymphocytes (CD4⁺ cells that belong to T-helper 1)that recognize various proteins and peptides in brain cells as antigensparticipate (Matsumoto, Y. et al., Clinical Immunology, 1997, Volume 29,pp. 1207-1212; Yamamura, Ryu, Brain and Nerves, 2001, Volume 53, pp.707-713). The antigens recognized by these encephalitogenicT-lymphocytes are diverse, and it is suggested that, for instance,myelin basic protein (MBP), proteolipid protein (PLP), myelin-associatedglycoprotein (MAG), glial fibrillary acidic protein (GFAP), and S-100β,as well as myelin oligodendrocyte glycoprotein (MOG), whoseencephalitogenicity is apparent, could be the antigens.

Since the cDNAs, that code for the peptides and polypeptides of thepresent invention from which the peptides were derived, were originatedfrom brain tumor cells, and since PBMC derived from an MS patient thatwere stimulated by these peptides recognized each corresponding peptideand enhanced the production of IFN-γ, these peptides are thought toparticipate in MS. In addition, since the present invention revealedthat immunoglobulins E (IgE) that recognize these peptides are presentin the blood of MS patients, it is possible that stimulation of mastcells by the IgE that recognizes these peptides is related to thepathologic formation of MS.

Several treatments for multiple sclerosis are currently being performed.For example, treatment by administration of interferon-P, which is aimedat suppressing the recurrence and progression via the immunomodulationmechanism, and steroid pulse therapy, which is aimed at treating throughanti-inflammatory action and induction of lymphoid cell death are used.Furthermore, in order to suppress the recurrence and progression of MS,a method, wherein immunological tolerance against antigens that arerecognized by the T-lymphocytes is induced by peptides that arerecognized by encephalitogenic T-lymphocytes or analog peptides thereof,in which a portion of the peptide is substituted with other amino acids,is examined in MS and experimental autoimmune encephalomyelitis (EAE)which is an animal model of MS (Yamamura, T., Clinical Immunology, 1997,Volume 29, pp. 1213-1218; Gaur, A., Science, 1992, Volume 258, pp.1491-1494). Immunological tolerance means a state where immunoreactivityagainst a specific antigen is absent. That is to say, if theimmunological tolerance induced in MS, which is one of the autoimmunediseases triggered as a result of the enhancement of the immune responseagainst an autoantigen, it is expected that MS can be suppressed fromprogressing, ameliorated, and prevented from recurring. The peptides ofthe present invention can be used to induce such immunologicaltolerance. That is to say, it is possible that the peptides of thepresent invention can be used in the prevention and/or treatment of MS.

Accordingly, the present invention provides polypeptides that are codedby the aforementioned genes obtained from KNS60 tumor cells. Preferably,these are one or more polypeptides having any one of the amino acidsequences set forth in SEQ ID NO: 14 to 18 in the Sequence Listing.Since these polypeptides are recognized by an HLA-A2-restrictedantigen-specific CTL, they can be used as tumor antigens that induceand/or activate the CTL. Furthermore, these polypeptides can be used asmaterials to obtain tumor antigen peptides by specifying the tumorantigen epitopes.

The peptides of the present invention can be obtained, for instance, bydesigning peptides conformable to the HLA-A2-binding motif based on theamino acid sequences of the aforementioned polypeptides, and thenselecting from the designed peptides that are recognized by CTL in anHLA-A2-restricted manner. The peptides may be those that bind to HLA-A2and are presented on the surface of the antigen-presenting cells, andthe peptides have the characteristics of a tumor antigen epitope that isrecognized by CTL, and is a peptide having 5 or more, preferably 7 ormore, more preferably 9 or 10, amino acid residues. Even more preferableis a peptide having any one of the amino acid sequences set forth in SEQID NO: 1 to 13 in the Sequence Listing. Since these peptides arerecognized by antigen-specific CTL in an HLA-A2-restricted manner, theycan be used to induce and/or activate HLA-A2-restricted antigen-specificCTL.

The aforementioned polypeptides or peptides may be used singly or usedin combination of two or more in order to induce and/or activate CTL.Since CTL are a plurality of cell groups that recognize variousantigens, it is recommended that preferably two or more of these be usedin combination.

In addition, polypeptides or peptides, which have one or more aminoacids with mutation(s), such as deletion, substitution, addition, orinsertion, are also included in the scope of the present invention.Preferable are polypeptides or peptides having such mutation(s) andwhich are recognized by CTL, for instance, at least by HLA-A2-restrictedCTL. Peptides having such mutation(s) may be those that exist naturally,or those in which one or more mutation(s) has been introduced. The meansfor introducing mutation(s), such as deletion, substitution, addition,or insertion, are known, and the method by Ulmer (Science, 1983, Volume219, p. 666 et seq.) can, for instance, be used. Seeing that fundamentalcharacteristics (physical properties, activity, or immunologicalactivity and the like) of these peptides are not changed by theintroduction of such mutation(s), reciprocal substitution among, forexample, homologous amino acids (polar amino acids, non-polar aminoacids, hydrophobic amino acids, hydrophilic amino acids, positivelycharged amino acids, negatively charged amino acids, aromatic aminoacids, and the like) is readily inferred. Furthermore, these usablepeptides can be altered to the extent that no significant functionalalteration is involved, such as modifying their constituent amino groupor carboxyl group and the like.

The polynucleotides of the present invention are polynucleotides havingthe nucleotide sequences that respectively code for the peptides havingany one of the amino acid sequences set forth in SEQ ID NO: 1 to 13 orthe polypeptides having any one of the amino acid sequences set forth inSEQ ID NO: 14 to 18 in the Sequence Listing, or complementary nucleotidesequences thereof. More preferable are polynucleotides having any one ofthe nucleotide sequences set forth in SEQ ID NO: 19 to 23 in theSequence Listing, or complementary nucleotide sequences thereof.Furthermore, the polynucleotides may have at least 15 or more,preferably 21 to 30 or more, nucleotide sequences corresponding to theregions that code for tumor antigen epitopes among the amino acidsequences of the polypeptides pertaining to the present invention, orcomplementary nucleotide sequences thereof. In the present invention,polynucleotides having complementary nucleotide sequences may bedesignated as complementary strands. Selection of such a usefulpolynucleotide and determination of the nucleotide sequence thereof canbe carried out, for example, by employing well-known protein expressionsystems to confirm the ability of the expressed peptide or polypeptideto induce and/or activate CTL.

Furthermore, polynucleotides that hybridize to the above-mentionedpolynucleotides under stringent conditions are also included in thescope of the present invention. If a DNA molecule is taken as arepresentative example of a polynucleotide molecule, “a DNA moleculethat hybridizes to a DNA molecule under stringent conditions” can beobtained by methods described in, for instance, Sambrook et al., eds.,Molecular Cloning: A Laboratory Manual, 1989, Cold-Spring HarborLaboratory Press, Cold-Spring Harbor, N.Y. Here, “hybridizing understringent conditions” means that, under conditions where, for instance,after heating in a solution of 6×SSC (final concentration of 150 mM NaCland 15 mM trisodium citrate), 0.5% SDS, and 50% formamide at 42° C., andwashing in a solution of 0.1×SSC and 0.5% SDS at 68° C., a positivehybridization signal is still observed.

When expressed in cells that possess HLA-A2, the aforementionedpolynucleotides can be recognized by HLA-A2-restricted andantigen-specific CTL and/or can induce the CTL. In addition, thepolynucleotides have a poly (A) structure at the 3′ end thereof;however, the number of poly (A) does not influence the site encoding theamino acids which act as tumor antigens, and therefore, there is noparticular restriction on the number of poly (A) that thepolynucleotides possess.

The aforementioned polynucleotides all provide genetic informationuseful in preparing the polypeptides or peptides of the presentinvention, or may be used as nucleic acid reagents or standards.

Recombinant vectors are obtained by integrating the aforementionedpolynucleotides into adequate vector DNAs. Vector DNAs to be used areselected appropriately depending on the host type and purpose of use.Vector DNAs may be those obtained by extracting vector DNAs naturallypresent, and also may be those that lack a part of the DNAs other thanthat required for multiplication. For instance, chromosome-, episome-,and virus-derived vectors; for instance bacterial plasmid-derived,bacteriophage-derived, transposon-derived, yeast episome-derived,insertion element-derived, yeast chromosomal element-derived;virus-derived vectors, such as from baculovirus, papovavirus, SV40,vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus, andretrovirus, as well as vectors combining these; vectors derived fromgenetic elements of plasmid and bacteriophage, such as, cosmid andphagemid and the like, may be exemplified. In addition, expressionvectors, cloning vectors, and the like may be used depending on thepurpose.

A recombinant vector has, as components, the target gene sequence andgene sequences that carry information regarding replication andregulation, such as a promoter, ribosome binding site, terminator,signal sequence, and enhancer. The vector may be created by combiningthe components by using methods well known in the art. As the method forintegrating the polynucleotides of the present invention into thepreviously mentioned vector DNAs, methods well known to one skilled inthe art may also be adopted. For instance, a method may be used in whichsuitable restriction endonucleases are selected; DNA is cleaved atspecific sites using these and then mixed with DNA to be used as avector, which has been treated in the same way, followed by relegatingwith a ligase. Otherwise, a desired recombinant vector may be obtainedby ligating an adequate linker to the target polynucleotide followed byinserting the resultant into a multi-cloning site of a vector suitablefor a purpose.

Transformants can be obtained by introducing the vector DNA, into whichthe aforementioned polynucleotide has been integrated, into a host thatis well-known to one skilled in the art by methods that are well-knownto one skilled in the art. Escherichia coli, yeast, Bacillus subtilis,insect cells, or animal cells may be cited as examples of hosts. Inpreferred systems, when carrying out transfection, the method ofintegration into chromosomes may be cited if gene stability is to beconsidered; however, for simplicity, auto-replicating systems that useextranuclear genes may be employed. Introduction of a vector DNA into ahost cell can be carried out by standard methods, for instance, asdescribed in Sambrook et al. eds., Molecular Cloning: A LaboratoryManual, Second Edition, Cold-Spring Harbor Laboratory Press, 1989.Concretely, calcium phosphate transfection, DEAE-dextran-mediatedtransfection, microinjection, cationic lipid-mediated transfection,electroporation, transduction, scrape loading, ballistic introduction,infection, and the like can be cited.

The polypeptides or peptides of the present invention can be provided ifan expression vector is used as a vector DNA to be introduced into theaforementioned transformant. The transformant having an expressionvector DNA, into which the aforementioned polynucleotide has beenintegrated, can be cultured under culture conditions that are optimalfor each host and well-known to one skilled in the art. The culture maybe performed using as an indicator the action of the polypeptides orpeptides of the present invention expressed by the transformant, forexample, the action of at least inducing and/or activating CTL; or thequantity of polypeptides or peptides generated inside or outside thehost; or a passage culture or a batch culture may be performed using thequantity of transformant in the culture medium.

The polypeptides or peptides of the present invention may bemanufactured by genetic engineering techniques as mentioned above, usingthe aforementioned vectors or transformants. In addition, they may alsobe manufactured by any method known in general peptide chemistry. Forinstance, methods described in Peptide Synthesis, 1975, Maruzen Co.,Ltd., or Peptide Synthesis, 1996, Interscience, New York can be used.Naturally, many known methods can be used.

In terms of purification and collection of the polypeptides or peptidesof the present invention, it is possible to purify and collect them byusing gel filtration chromatography, ion column chromatography, oraffinity chromatography and the like, or combinations thereof, or by afractionation means based on a difference in solubility using ammoniumsulfate or alcohol and the like, using their characteristics andbiological activity as indicators, for instance whether or not thepeptides are at least recognized by CTL and/or induce CTL. Morepreferably a method can be used, in which polypeptides or peptides arespecifically adsorbed and collected by using polyclonal antibodies ormonoclonal antibodies, which can be prepared against the polypeptides orthe peptides based on the information of their amino acid sequences.

The antibodies can be created using the aforementioned polypeptides orpeptides as antigens. The antigen can be the polypeptide or peptide, orfragments thereof, and can be constituted by at least eight, preferablyat least ten, more preferably at least twelve, even more preferablyfifteen or more amino acids. In order to create antibodies that arespecific to the polypeptides and/or peptides, it is preferable to useregions having amino acid sequences that are unique to the polypeptidesor peptides. This amino acid sequence does not necessarily need to behomologous to the amino acid sequences of the polypeptides or peptides.Sites that are exposed to the exterior of the tertiary structure of thepolypeptides or peptides are preferred. Even if the amino acid sequencesof the exposed sites are discontinuous in the primary structure, itsuffices that they be amino acid sequences that are continuous on theexposed site. There are no particular limits on the antibodies, as longas they immunologically bind to or recognize the polypeptides and/orpeptides. Whether or not this binding or recognition occurs isdetermined by the well-known antigen-antibody-binding reaction.

Any antibody preparation method well known in the art can be used toproduce the antibodies. For instance, they can be obtained byadministering to an animal a polypeptide or peptide of the presentinvention with or without linking such to a carrier, in the presence orabsence of an adjuvant, to induce immunity, such as a humoral responseand/or cell-mediated response. The carrier is not limited in particular,as long as it does not exert a harmful effect by itself on the host andis capable of enhancing antigenicity; for example, cellulose, polymericamino acids, albumin, key hole limpet hemocyanin (KLH), and the like.Examples of the adjuvant can be a Freund complete adjuvant (FcA), Freundincomplete adjuvant (FIA), Ribi (MPL), Ribi (TDM), Ribi (MPL+TDM),Bordetella pertussis vaccine, muramyl dipeptide (MDP), aluminum adjuvant(ALUM), and combinations thereof. For animals to be immunized, mouse,rat, rabbit, goat, horse, and the like may be suitably used.

The polyclonal antibody is obtained from the serum of the animalssubjected to the aforementioned immunization means, by any suitablemethod for collecting antibodies. As a preferable means, theimmuno-affinity chromatography method can be cited.

The monoclonal antibody can be produced by collecting antibody-producingcells (for example, a lymphocyte derived from a spleen or a lymph node)from the animals subjected to the aforementioned immunization, followedby introducing a well-known transformation technique with a permanentlyproliferating cell (for example, myeloma strain such as P3X63Ag8 cells.)For example, an antibody-producing cell can be fused with a permanentlyproliferating cell by methods that are well-known to one skilled in theart to create a hybridoma, which can then be cloned, followed byselecting hybridoma producing an antibody that recognizes specificallythe aforementioned polypeptides and/or peptides. The antibody can thenbe collected from a culture solution of the hybridoma.

The polyclonal antibody or monoclonal antibody thus obtained, which canrecognize and bind to the aforementioned polypeptides and/or peptides,can be used as a purification antibody, reagent, or labeling marker forthe polypeptides or the peptides.

The aforementioned polypeptides or peptides, polynucleotides codingtherefor, complementary strands thereof, cells that have beentransformed based on the information of the amino acid sequence andnucleotide sequence thereof, or antibodies that immunologicallyrecognize the above, provide, independently or in combination, aneffective means of identifying a substance that may enhance recognitionof the polypeptide or peptide by CTL. The identification method of thepresent invention can be established using pharmaceutical screeningsystems that are well-known in the art. For instance, a substance thatenhances recognition of the polypeptides or peptides of the presentinvention by CTL can be screened by stimulating CTL withantigen-presenting cells which have been pulsed with a tumor antigenpeptide, or antigen-presenting cells in which a tumor antigen isexpressed, followed by establishing an experimental system to measurerecognition of the tumor antigen peptide or the tumor antigen by the CTLand/or activation of the CTL, and examining the test substance.Antigen-presenting cells can include cells that retain HLA-A2, forexample, cell strains that retain HLA-A2, or more specifically, T2 cellsand the like. Or, even cells that do not possess HLA-A2 can be used bygenetically introducing HLA-A2 cDNA so that an HLA-A2 molecule isexpressed on the cell surface. Pulsing the tumor antigen toantigen-presenting cells can be performed by co-culturingantigen-presenting cells and tumor antigen with any suitable method. Thetumor antigen can be expressed in antigen-presenting cells byintroducing the polynucleotide that codes for the antigen into the cellby any suitable technique. Examples of CTL include HLA-A2-restricted CTLstrain or HLA-A2-restricted CTL line, such as OK-CTL and the like.Recognition of the tumor antigen peptide or the tumor antigen by the CTLand/or activation of the CTL can be easily determined by measuring IFN-γproduction from the CTL. This experimental system is to describe oneidentification method, and the identification method of the presentinvention is not restricted thereby.

The present invention also includes the compound obtained by theaforementioned identification. The compound may be a compound thatinteracts with a polypeptide or a peptide of the present invention, forexample, a peptide having the amino acid sequence of any one of those ofSEQ ID NO:1 to SEQ ID NO:13 in the Sequence Listing, or a polypeptidehaving the amino acid sequence of any one of those of SEQ ID NO:14 toSEQ ID NO:18 in the Sequence Listing, and/or an HLA-A2 to enhancerecognition of the polypeptide or the peptide by HLA-A2-restricted CTL.In addition, a compound or the like that interacts with a polynucleotideof the present invention and enhances expression thereof is also withinthe scope of the present invention. A compound screened in such a mannercan be prepared as a pharmaceutical composition by selecting it whiletaking into account the balance between biological usefulness andtoxicity.

The polypeptides or peptides provided in the present invention can beused as tumor antigens or tumor antigen peptides, in order to induceand/or activate CTL that are antigen-specific in an HLA-A2-restrictedmanner. That is to say, medicaments can be used that contain one or morepolypeptides selected from the aforementioned polypeptides and/or one ormore peptides selected from the aforementioned peptides. Methods forinducing CTL which are characterized by the use of one or morepolypeptides/peptides selected from the aforementionedpolypeptides/peptides, as well as agents for inducing CTL which cancontain one or more polypeptides/peptides selected from theaforementioned polypeptides/peptides, are also included within the scopeof the present invention. The aforementioned method for inducing CTL caninclude, as one embodiment thereof, a step of pulsing a peptide of thepresent invention to an antigen-presenting cell, or a step of having apolypeptide of the present invention expressed in the antigen-presentingcells. A further step may include stimulating a cell group that containsCTL precursor cells, using the antigen-presenting cells obtained by anyone of the aforementioned steps. Examples of antigen-presenting cellsinclude cells that retain HLA-A2, such as cell strains that retainHLA-A2, more concretely, T2 cells and the like. Furthermore, cells thatordinary do not possess HLA-A2 can be used by genetically introducingHLA-A2 cDNA to make them express an HLA-A2 molecule on the cell surface.Pulsing the peptide to antigen-presenting cells can be performed byco-culturing antigen-presenting cells and tumor antigen by any suitablemethod. The polypeptide can be expressed in the cells by introducing thepolynucleotide that codes therefor into an antigen-presenting cell byway of a general genetic engineering technique. Cell groups that containCTL precursor cells are, for instance, peripheral blood cells, or morepreferably, peripheral blood mononuclear cells.

In addition, a pharmaceutical composition can be provided which containsan effective dose of at least one of the following: the polypeptides orthe peptides of the present invention; polynucleotides coding for thepolypeptides and complementary strands thereof; recombinant vectorscreated based on the information of the amino acid sequences andnucleotide sequences thereof; cells that are transformed by therecombinant vectors; antibodies that immunologically recognize thepolypeptides/peptides; compounds that interact with the polypeptides orthe peptides, and/or, HLA-A2, and enhance recognition of thepolypeptides or peptides by CTL; or compounds that interact with thepolynucleotides and enhance expression thereof, when used alone or incombination of a plurality thereof. For instance, the pharmaceuticalcomposition could be useful in the treatment of cancers, such as thetreatment of brain tumors. Considering that the HLA-A2 allele is foundin approximately 40% of Japanese, approximately 53% of Chinese,approximately 49% of North American Caucasians, approximately 38% ofSouth American Caucasians, and approximately 23% of African Blacks, thepharmaceutical composition of the present invention can be effective ona multitude of patients.

Concretely, medicaments containing, for instance, one or morepolypeptides/peptides selected from the aforementionedpolypeptides/peptides and pharmaceutical compositions that containeffective doses of one or more polypeptides selected from theaforementioned polypeptides and/or one or more peptides selected fromthe aforementioned peptides, can be used as so-called cancer vaccines.The term cancer vaccine as used herein means a drug that selectivelydamages tumor cells by inducing and/or enhancing a specific immuneresponse against the tumor cells. The dosage thereof can be determinedwith appropriate modifications, according to the extent of recognitionof the polypeptides or peptides by CTL. For example, in general it isbetween 0.01 and 100 mg/day/adult human, or preferably 0.1 and 10mg/day/adult human as an active principle. This can be administered onceevery few days to every few months. Administration may be carried outaccording to well-known methods for administrating a peptide for medicaluse, preferably subcutaneously, intravenously, or intramuscularly. Inorder to induce and/or enhance the immune response duringadministration, the polypeptide and/or the peptide of the presentinvention may be used with or without linking such to a carrier in thepresence or absence of an appropriate adjuvant. The carrier is notlimited in particular, as long as it exerts no harmful effect by itselfon the human body and is capable of enhancing antigenicity; cellulose,polymeric amino acids, albumin, and the like can be given as examples.Adjuvants may be those used in general for peptide vaccine inoculation,and a Freund incomplete adjuvant (FIA), aluminum adjuvant (ALUM),Bordetella pertussis vaccine, mineral oil, and the like can be given asexamples. In addition, the formulation can be suitably selected byapplying a suitable well known method for formulating a peptide.

Otherwise, an effective cancer vaccine effect can be obtained also by,with the aforementioned polypeptide or peptide, inducing and/oractivating CTL in the mononuclear cell fraction collected from theperipheral blood of a patient and then returning the fraction ofinterest into the blood of the patient. Culture conditions, such asmononuclear cell concentration, polypeptide or peptide concentration,culture time, and the like, can be determined by simply repeatingexperiments. A substance with ability to enhance the growth oflymphocytes, such as interleukin-2, may also be added during culturing.

The aforementioned polypeptides or peptides can be effectively usedalone or in combination as a cancer vaccine. Since it has been reportedthat multi-peptides based immunotherapies are effective (Non-PatentReferences 9, 10, and 11), and since the CTL of a cancer patient is agroup of cells that recognize a plurality of tumor antigens, rather thanusing one type of polypeptide or one type of peptide as a cancervaccine, higher effectiveness may be obtained by using a plurality oftypes in combination for use as a cancer vaccine.

The polynucleotides coding for the polypeptides or peptides of thepresent invention, or more preferably coding for the peptides, andcomplementary strands thereof, are useful in gene therapy of cancers;and brain tumors, for instance. There is a method in which vectorscontaining these polynucleotides can be directly introduced to the body.Furthermore, there is a method in which, after collecting cells fromhumans, the vectors can be extracorporeally introduced. Retrovirus,adenovirus, vaccinia virus, and the like are known as vectors, however,the retroviral system is recommended. For viruses, nonreplicative onescan be used. The dosage can be determined by adding appropriatemodifications according to the extent of recognition of the polypeptidesor peptides by the CTL, in general, it can be between 0.1 μg and 100mg/day/adult human, or preferably between 1 μg and 50 mg/day/adult humanas the content of DNAs that code for the polypeptides or peptides of thepresent invention.

In addition, the polypeptides and the peptides of the present inventionwere identified from a cDNA library derived from brain tumor cells, andthe PBMC which was derived from a multiple sclerosis (MS) patient andstimulated by these peptides, for example, by the peptides having anyone of the amino acid sequences listed as SEQ ID NO: 1 to 6 in theSequence Listing, recognized each corresponding peptide and enhancedIFN-γ production. Furthermore, IgE which recognizes these peptides wasdetected in the serum of MS patients. These indicate that there is apossibility of these polypeptides and peptides participating in thepathologic formation of MS. Therefore, by using these polypeptides orpeptides, preferably peptides, it is possible to prevent and/or treatMS.

For instance, by using the peptides of the present invention andinducing immunological tolerance in MS patients, it is possible toinhibit the progression of, ameliorate, and prevent the recurrence ofMS. The dosage, administration period, and administration method of thepeptides, as well as the form of administration are adapted forinduction of immunological tolerance, and can be determined according toprescriptions generally applied for an antigen peptide therapy againstautoimmune diseases. Furthermore, necessary modifications can beperformed depending on the degree of seriousness and the like of theparticular pathology. Generally, in order to induce immunologicaltolerance by peptides, it is necessary to administer in largequantities; for instance, between 0.01 and 100 mg/day/human. Theadministration method should be performed similarly to the well-knownadministration methods for medically used peptides, and preferably, itis performed via subcutaneous administration, intravascularadministration, intranasal administration, or intramuscularadministration. During administration, the peptide may be used singly,or may be used together with a suitable well-known adjuvant. For theinduction of immunological tolerance, a single peptide may be used, or aplurality of peptides may be used in combination. In addition, analogpeptides resulting from the introduction of one to several amino acidsubstitutions in a peptide, acylated peptides, or peptides bound to MHCclass II molecules and the like may be used to increase the effect ofimmunological tolerance induction.

In addition, the polypeptides of the present invention, for instance,polypeptides having an amino acid sequence listed as SEQ ID NO: 14 or 15of the Sequence Listing, can also be used in the prevention and/ortreatment of MS. Furthermore, it is possible to prevent and/or treat MSby inserting the polynucleotides that code for the peptides orpolypeptides of the present invention and complementary strands thereofinto a vector, and expressing the peptides or polypeptides in cells orin vivo. In addition, the aforementioned pharmaceutical composition canbe used in the prevention and/or treatment of MS.

The aforementioned polypeptide or peptide, polynucleotide that codes forthe polypeptide or the peptide, and the complementary strand thereof, aswell as the antibody that immunologically recognizes the polypeptide orthe peptide, can be used singly as a diagnostic marker, reagent, and thelike. When used as reagents, they may contain a substance, such as abuffering solution, salt, stabilization agent, and/or antiseptic agent.In addition, the present invention also provides a reagent kitcomprising one or more containers that are filled with one or more kindsof these reagents. Furthermore, for formulations, suitable formulationmeans can be used that are well-known for peptides, polypeptides,polynucleotides, antibodies, and the like.

The abovementioned reagents and reagent kits can be used in theaforementioned screening methods of the present invention. In addition,they can be used for quantitatively and/or qualitatively measuring thepolypeptides or peptides of the present invention, or polynucleotidescoding for any one thereof. This measuring method can be establishedusing methods that are well-known to those skilled in the art. Asexamples of methods that can be used, a radio immunoassay, competitivebinding assay, Western blot analysis, ELISA, and the like can be given.In addition, in terms of nucleic acids, it is possible to detect andquantify them at the RNA level using, for instance, amplification, PCR,RT-PCR, RNase protection, Northern blotting, and other hybridizationmethods.

The aforementioned reagents, reagent kits, and measurement methods canbe used in the detection method of diseases related to the expression oractivation of the polypeptides or peptides of the present invention. Asexamples of diseases of interest, cancer diseases and the like, morepreferably cancer diseases in which the cancer is HLA-A2 positive, evenmore preferably brain tumors, can be given.

As examples of samples to be measured, cells derived from individuals,for instance, blood, urine, saliva, spinal fluid, tissue biopsy, ornecropsy materials and the like, can be given. In addition, the nucleicacids to be measured can be obtained from each of the aforementionedsamples by nucleic acid preparation methods that are well-known in theart. For the nucleic acid, genomic DNA can be used directly fordetection, or it may be enzymatically amplified prior to analysis by PCRor other amplification methods. RNA or cDNA may be used in a similarmanner. In addition, deletions and insertions can be detected by changesin the sizes of the amplification products, in comparison with a normalgenotype. In addition, point mutations can be identified by hybridizingthe amplified DNA to a labeled DNA that codes for the aforementionedpolypeptide.

With a sample derived from an individual, a disease can be detected, forinstance, by detecting the presence of the corresponding nucleic acid tothe polynucleotide that codes for the polypeptide of interest; bydetermining the quantity of the nucleic acid; and/or identifying themutation of the nucleic acid. This nucleic acid can be detected by usingits interaction with, and its responsiveness to, the polynucleotide thatcodes for the polypeptide of interest. Furthermore, a disease can bedetected by determining the in vivo distribution of the polypeptide orpeptide in the individual; by detecting the presence of the polypeptideor peptide; and/or by determining the quantity of the polypeptide orpeptide; or by detecting the mutation of the polypeptide or peptide.

Furthermore, the aforementioned diseases can be examined and diagnosedby qualitatively or quantitatively measuring the polypeptides orpeptides of the present invention, or the nucleic acid coding therefor,as diagnostics markers. That is to say, by using the afore-mentioneddetection method, a method for examining or diagnosing the diseases canfurther be performed.

EXAMPLES

The present invention will be described more concretely in thefollowing, by way of examples; however, the present invention is notlimited to these examples.

Example 1 Establishment of HLA-A2-Restricted CTL

An HLA-A2-restricted tumor-specific cytotoxic T-lymphocyte strain wasestablished from tumor-infiltrating lymphocytes (TIL) of a colon cancerpatient (HLA-A0207/3101, HLA-B46/51, HLA-Cw1), according to a methoddescribed in the literature (International Journal of Cancer, 1999,Volume 81, pp. 459-466; Non-Patent Reference 4). First, TIL obtainedfrom a colon cancer patient were cultured for more than 50 days with theaddition of 100 U/ml of recombinant human interleukin-2 (IL-2). Every 7culture days, a portion of these IL-2-activated TIL were collected,co-cultured with various tumor cells or normal cells, and the CTLactivity thereof was assayed by measuring the production of IFN-γ andwith a test for Cr⁵¹ released from the tumor cells (Non-Patent Reference4). Measurement of IFN-γ was performed by enzyme-linked immunosorbentassay (ELISA). At day 58 of culturing, OK-CTL was obtained, which is asubline that demonstrates a tumor-specific cytotoxic activity in anHLA-A2-restricted manner. The phenotype was CD3⁺CD4⁻CD8⁺ for 80% of theOK-CTL, the phenotype of the remaining 20% being CD3⁺CD4⁺CD8⁻.

OK-CTL recognized HLA-A0201⁺ pancreatic adenocarcinoma cell Panc-1,HLA-A0201⁺ colon adenocarcinoma cell SW620, HLA-A0206⁺ esophagealsquamous cell carcinoma (SCC) cell KE3, HLA-A0207⁺ oral SCC cell CA9-22,HLA-A2⁺ astrocytoma cell U251, and HLA-A2⁺ glioma cell KNS60, andproduced IFN-γ. In addition, it demonstrated sufficient cytotoxicactivity. However, it did not demonstrate cytotoxic activity againstHLA-A2⁻ tumor cells, autologous Epstein-Barr virus (EBV)-transformedB-cell (hereinafter may be abbreviated as EB-BC), and autologousphytohaemagglutinin (PHA) blastoid T-lymphocyte (Autologous PHA-blastoidT-lymphocytes; hereinafter may be abbreviated as PHA-blast). Inaddition, OK-CTL lysed all the HLA-A2⁺ tumor cells (HLA-A0201⁺ breastadenocarcinoma cell R27, primary hepatocellular carcinoma cell HAK-2,melanoma cell SK-MEL-5 and astrocytoma cell SF126; HLA-A0206+pulmonaryadenocarcinoma cell PC9, as well as pulmonary adenocarcinoma cell 1-87;and HLA-A0207⁺ cervical SCC cell OMC-4) that were examined. Recognitionof these cells by OK-CTL and the resulting production of IFN-γ wereinhibited by anti-HLA class I monoclonal antibody (mAb), anti-CD8 mAb,or anti-HLA-A2 mAb, but were not inhibited by other mAbs. This showsthat OK-CTL recognizes tumor cells in an HLA-A2-restricted manner anddemonstrates cytotoxic activity.

Example 2 Isolation and Identification of cDNA Clones Coding for TumorAntigens

The genes coding for tumor antigens that were recognized by OK-CTLobtained in Example 1 were isolated and identified from a cDNA libraryof glioma cell KNS60 that was derived from human brain tumor cellsaccording to a known method (Journal of Experimental Medicine, 1998,Volume 187, pp. 277-288). First, the poly (A)⁺ RNA from KNS60 tumorcells (deposited as IF050357 at the Institute for Fermentation) wasprepared according to methods in the art. The poly (A)⁺ RNA obtained wasconverted into cDNA, ligated to a Sal I adapter, and inserted into theexpression vector pCMV-SPORT-2 (Invitrogen). In addition, each of thecDNAs of HLA-A0207, HLA-A2402, and HLA-A2601 were amplified by reversetranscription polymerase chain reaction (hereinafter abbreviated as PCR)and cloned into the eucaryotic cell expression vector pCR3 (Invitrogen).

The cDNA clones obtained from the KNS60 tumor cells were pooled intopools of 100 clones each, and then 100 ng of the cDNA pooled in eachwell of a U-shaped 96-well plate, and 100 ng of HLA-A0207 cDNA,HLA-A2402 cDNA, or HLA-A2601 cDNA were incubated in 100 μl of a 1:200mixture of lipofectoamine/Opti-MEM (Invitrogen) for 30 minutes. Anamount of 50 μl from this mixture was added to COS-7 cells (1×10⁴) andincubated for 6 hours in a U-shaped 96-well plate for co-transfection.Next, RPMI-1640 culture medium containing 10% FCS was added and culturedfor 2 days, and OK-CTL (2×10⁵) were added into each well. Afterincubating for an additional 18 hours, 100 μl of supernatant wascollected, and the IFN-γ produced was measured by ELISA, so as to screenthe pools of the cDNA library. At this time COS-7 cells in which no genewas introduced was used as target cells for a negative control, and theproduction of IFN-γ by OK-CTL was examined. The value of IFN-γ producedwas subtracted from each measurement value as the background.

After confirming reproducibility of the pools from the cDNA library ofKNS60 tumor cells that enhanced production of IFN-γ from CTL, cloneswere individually taken up from each cDNA pool whose reproducibility hadbeen verified, and screening was performed by the same method aspreviously described, so as to select clones derived from independentpools that were recognized by CTL. Furthermore, the dose dependency ofthe clones obtained was verified by the same method as previouslydescribed, and five types of clone were finally obtained. When each ofthese five types of cDNA clone was co-transfected with the HLA-A0207cDNA into COS-7 cells, these clones were recognized by OK-CTL andenhanced production of IFN-γ from OK-CTL in a dose-dependent manner.However, when these cDNA clones were co-transfected with HLA-A2402 cDNAor HLA-A2601 cDNA, no enhancement of IFN-γ production by OK-CTL wasobserved. This makes it clear that these five types of cDNA clone codefor tumor antigens that are recognized by OK-CTL in an HLA-A2-restrictedmanner. The results for clone 8B6 and clone 2G2 are shown in FIG. 1 andFIG. 2, respectively, as representative examples. The same results wereobtained for the other clones.

Determination of the nucleotide sequences of the aforementioned fivetypes of cDNA clones, that is, clone 8B6, clone 2G2, clone 4G3, clone7H9, and clone 1B10, was carried out by the dideoxynucleotide sequencingmethod using a DNA sequencing kit (Perkin-Elmer) and the ABIPRISM® 377DNA Sequencer (Perkin-Elmer), (SEQ ID NO: 19 to 23). Furthermore, theamino acid sequence (SEQ ID NO: 14 to 18) encoded by each cDNA clone wasdeduced from the nucleotide sequence. In addition, a homology search wascarried out against the GenBank for the nucleotide sequence of eachclone obtained. These results are shown in Table 1 mentioned above.

Example 3 Peptide Preparation and CTL Activation Test

Tumor antigen peptides were obtained from the genes isolated andidentified in Example 2 that code for tumor antigens. First, using acomputer, a search for motifs that may bind to the HLA-A2 molecule wascarried out for the amino acid sequences coded by each of these genes,or for those coded by genes that are highly homologous to these genes<http://bimas.dcrt.nih.gov//molbio/hla_bind/>. Based on the results,differing 9-mer or 10-mer peptides were designed and synthesized by amethod well known in the art. Peptides having a purity of 70% or morewere obtained thereby.

Each of the synthesized peptides (0.001 μM to 30 μM or 0.1 ng/ml to 100μg/ml) was incubated with T2 cells expressing HLA-A2 molecules on thecell surface in a form that does not bind to peptides (Cancer Research,1994, Volume 54, pp. 1071-1076) for 2 hours at 37° C. under conditionsof 5% CO₂-95% air, to make the peptide of interest bind to the HLA-A2molecule expressed on the cell surface. The T2 cells, pulsed with eachpeptide in this way, were used as the target cells (T). In addition, theOK-CTL obtained in Example 1 were used as effector cells (E). Targetcells amounting to 1×10⁴ and effector cells amounting to 1×10⁵ weremixed (E/T ratio=10) and incubated for 18 hours. After incubation, 100μl of supernatant was collected, and IFN-γ was measured by ELISA. Thequantity of IFN-γ produced by CTL against T2 cells that were not pulsedwith a peptide was taken as the background and subtracted from eachmeasurement value.

As a result, the 13 types of peptide were respectively recognized byOK-CTL in a dose-dependent manner and enhanced production of IFN-γ fromOK-CTL. These peptides are P101 (SEQ ID NO: 1), P102 (SEQ ID NO: 2), andP103 (SEQ ID NO: 3) derived from clone 8B6; P104 (SEQ ID NO: 4), P105(SEQ ID NO: 5), and P106 (SEQ ID NO: 6) derived from clone 2G2; P1 (SEQID NO: 7), P2 (SEQ ID NO: 8), and P3 (SEQ ID NO: 9) derived from clone4G3; P6 (SEQ ID NO: 10) derived from clone 7H9; as well as P14 (SEQ IDNO: 11), P18 (SEQ ID NO: 12), and P19 (SEQ ID NO: 13) derived from clone1B10. The results for the three types of peptide derived from clone 8B6and the three types of peptide derived from clone 2G2 are shown in FIG.3 and FIG. 4, respectively, and the results for the seven types ofpeptide derived from clone 4G3, clone 7H9, and clone 1B10 are shown inFIG. 5. In FIG. 5, the peptides that were recognized by CTL and/orinduced CTL are shown by the solid lines. In addition, the peptidederived from human immunodeficiency virus (hereinafter abbreviated asHIV), which was used as a negative control, is a peptide that may bindto the HLA-A2 molecule. It was not recognized by CTL and did not enhanceproduction of IFN-γ by CTL.

Example 4 Induction of CTL by Peptides from Peripheral Blood MononuclearCells of Cancer Patients

Among the 13 types of peptide obtained in Example 3, three peptides,P101 (SEQ ID NO: 1), P102 (SEQ ID NO: 2), and P103 (SEQ ID NO: 3), whichwere derived from clone 8B6, as well as three peptides, P104 (SEQ ID NO:4), P105 (SEQ ID NO: 5), and P106 (SEQ ID NO: 6), which were derivedfrom clone 2G2, were examined for their ability to induceHLA-A2-restricted CTL from PBMC obtained from an HLA-A2⁺ brain tumorpatient or healthy subject.

PBMC were prepared by methods of the art from the blood of a metastaticbrain tumor patient (hereinafter referred to as Patient Case 1), ameningioma patient (hereinafter referred to as Patient Case 2), or 2healthy subjects. The PBMC amounting to 1×10⁵ were respectivelyincubated with 10 μM of each peptide in each well of a 96-well U-shapedmicroculture plate (Nunc) to which 200 μl of a culture medium(consisting of 45% RPMI-1640 culture medium, 45% AIM-V® culture medium(Invitrogen), 100 U/ml of IL-2, 0.1 mM of MEM non-essential amino acidsolution (Invitrogen), and 10% fetal calf serum (FCS)) had been added.At 4 days and 7 days of culturing, half the culture medium was removedand replaced with a culture medium of the composition containing thecorresponding peptide described above.

At 10 days culture, the cells were collected, washed, and used aseffector cells. T2 cells were pulsed with each corresponding peptide,and used as target cells. Both cells were mixed and cultured by the samemethod as in Example 3, and the quantity of IFN-γ produced was measured.In so doing, the effector cells were paired with the target cells insuch a way that the peptide used to induce the effector cells and thepeptide pulsed to T2 cells were the same. The results for Patient Case 1and Patient Case 2 are shown in FIG. 6A and FIG. 6B, respectively. InPatient Case 1, the three peptides, P101 (SEQ ID NO: 1), P103 (SEQ IDNO: 3), and P106 (SEQ ID NO: 6) enhanced production of IFN-γ from PBMC.Furthermore, P104 (SEQ ID NO: 4) in particular enhanced production ofIFN-γ in Patient Case 2. These peptides induced HLA-A2-restricted CTLfrom the PBMC of brain tumor patients. However, neither peptide inducedCTL from the PBMC of healthy subjects. Meanwhile, the peptides havingthe HLA-A2-binding motif, which were derived from an influenza virus(hereinafter may be abbreviated as flu) and used as positive controls,enhanced production of IFN-γ from PBMC in Patient Case 1 withsignificance. No significance was observed in the enhancement ofproduction of IFN-γ from PBMC in Patient Case 2. Thus, the fact thatthere are peptides that do not induce CTL from PBMC in brain tumorcancer patients when cultured under the aforementioned conditions, eventhough the peptides can activate OK-CTL, and the fact that there arecases in which CTL are induced and cases in which CTL are not induced,even though the same peptide is used, may be attributed to CTLprecursors present in the peripheral blood of cancer patients. Theseprecursors being a plurality of cell groups capable of recognizing avariety of antigens. Therefore, even if a peptide is not capable ofinducing CTL from the PBMC of the brain tumor patient, despite beingrecognized by OK-CTL and enhancing IFN-γ production thereby, there isstill ample possibility of inducing CTL in another patient.

Furthermore, the cells stimulated with the peptide and cultured for 10days were cultured for a further 10 days, and then the cytotoxicactivity against various target cells (HLA-A2⁺) was measured by astandard 6-hour ⁵¹Cr release test. The measurement was carried out byvarying the effector cell/target cell (E/T) ratio; the results obtainedwere expressed as the percentage of specific lysis. KNS60 tumor cells,KALS-1 tumor cells, autologous Epstein-Barr transformed B cells (EB-BC),and PHA-blasts were used as target cells.

The results were that all of the PBMC derived from Patient Case 1 whoseIFN-γ production was accelerated by P101 (SEQ ID NO: 1), P103 (SEQ IDNO: 3), or P106 (SEQ ID NO: 6), specifically lysed KNS60 tumor cells(FIG. 7A, FIG. 7B, and FIG. 7C, respectively). In addition, theaforementioned PBMC derived from Patient Case 2 whose IFN-γ productionwas accelerated by P104 (SEQ ID NO: 4) also specifically lysed KNS60tumor cells (FIG. 7D). This makes it clear that the aforementionedpeptide can induce HLA-A2-restricted tumor-specific cytotoxicT-lymphocytes from the PBMC of cancer patients.

Example 5 Induction of CTL by Peptides from Peripheral Blood MononuclearCells of Cancer Patients

Among the 13 types of peptide obtained in Example 3, three peptides,P101 (SEQ ID NO: 1), P102 (SEQ ID NO: 2), and P103 (SEQ ID NO: 3), whichwere derived from clone 8B6, as well as three peptides, P104 (SEQ ID NO:4), P105 (SEQ ID NO: 5), and P106 (SEQ ID NO: 6), which were derivedfrom clone 2G2, were examined for their ability to induce CTL in anHLA-A2-restricted manner from PBMC obtained from HLA-A2⁺brain tumorpatients.

The PBMC were prepared by methods of the art from the blood of threeprimary brain tumor patients and one metastatic brain tumor patient. ThePBMC obtained were stimulated by incubation with each peptide in thesame way as in Example 4, and were cultured by the same method as inExample 3 using T2 cells that were pulsed with each correspondingpeptide or KNS60 tumor cells as target cells, followed by measuring thequantity of IFN-γ produced. When T2 cells that were pulsed with thepeptides were used as target cells, the PBMC was paired with the T2cells in such a way that the peptide used to stimulate PBMC and thepeptide pulsed to T2 cells were the same.

Representative results for P101 (SEQ ID NO: 1), P102 (SEQ ID NO: 2), andP103 (SEQ ID NO: 3) are shown in FIG. 8, and representative results forP104 (SEQ ID NO: 4), P105 (SEQ ID NO: 5), and P106 (SEQ ID NO: 6) areshown in FIG. 9. The PBMC that were stimulated by P101 (SEQ ID NO: 1)and P103 (SEQ ID NO: 3) in Patient Case 3 (FIG. 8A), by P102 (SEQ ID NO:2) and P103 (SEQ ID NO: 3) in Patient Case 4 (FIG. 8B), as well as byP104 (SEQ ID NO: 4) and P106 (SEQ ID NO: 6) in Patient Case 5 (FIG. 9)recognized T2 cells that were pulsed with each corresponding peptideand/or HLA-A2+tumor cells, and enhanced production of IFN-γ. That is tosay, these peptides induced HLA-A2-restricted CTL from the PBMC of braintumor patients.

Example 6 Peptide Recognition in Peripheral Blood Mononuclear Cells ofMultiple Sclerosis Patients

Among the 13 types of peptide obtained in Example 3, three peptides,P101 (SEQ ID NO: 1), P102 (SEQ ID NO: 2), and P103 (SEQ ID NO: 3), whichwere derived from clone 8B6, as well as three peptides, P104 (SEQ ID NO:4), P105 (SEQ ID NO: 5), and P106 (SEQ ID NO: 6), which were derivedfrom clone 2G2, were examined for their ability to induce CTL from PBMCobtained from multiple sclerosis patients having HLA-A2 as the HLA typeand healthy subjects, in the same manner as in Example 5.

Representative results for P101 (SEQ ID NO: 1), P102 (SEQ ID NO: 2), andP103 (SEQ ID NO: 3) are shown in FIG. 10, and representative results forP104 (SEQ ID NO: 4), P105 (SEQ ID NO: 5), and P106 (SEQ ID NO: 6) areshown in FIG. 11. The PBMC that were stimulated by P103 (SEQ ID NO: 3)in MS Patient Case 6 (FIG. 10A), by P101 (SEQ ID NO: 1), P102 (SEQ IDNO: 2), and P103 (SEQ ID NO: 3) in MS Patient Case 7 (FIG. 10B), as wellas by P104 (SEQ ID NO: 4), P105 (SEQ ID NO: 5), and by P106 (SEQ ID NO:6) in MS Patient Case 8 (FIG. 11) recognized T2 cells that were pulsedwith each corresponding peptide and enhanced production of IFN-γ.However, HLA-A2⁺ tumor cells were not recognized, and production ofIFN-γ was not observed. In addition, the stimulated PBMC did not showcytotoxic activity against the aforementioned tumor cells. The sameresults were obtained for the PBMC obtained from the healthy subjects.

Multiple sclerosis is a representative disease associated withdemyelination in the central nervous system. The onset mechanism thereofhas not yet been elucidated; however, it is said to be an autoimmunedisease in which encephalitogenic T-lymphocytes participate. These Tlymphocytes recognize a variety of proteins and peptides that arepresent in brain cells as antigens, such as, for instance, MBP, PLP,MOG, MAG, or S-100β. Given that the peptides that were used in thepresent example are coded for by genes that were derived from a braintumor, and given that PBMC derived from MS patients that were stimulatedwith these peptides recognized each corresponding peptide and enhancedproduction of IFN-γ, there is a possibility that these peptides and thepolypeptides from which the peptides were derived participate in MS.

Example 7 Analysis of Cells Induced from the Peripheral BloodMononuclear Cells of a Multiple Sclerosis Patient by Peptides

Using P101 (SEQ ID NO: 1), P102 (SEQ ID NO: 2), P103 (SEQ ID NO: 3),P104 (SEQ ID NO: 4), P105 (SEQ ID NO: 5), and P106 (SEQ ID NO: 6), cellsthat can recognize these peptides were induced from PBMC that wereobtained from the multiple sclerosis patient and the healthy subject inExample 6. Next, an analysis of these cells was attempted.

First, PBMC from the healthy subject were cultured and stimulated withP104 (SEQ ID NO: 4) or P106 (SEQ ID NO: 6) in the same way as in Example5. The cells that had been stimulated with peptides recognized T2 cellsthat were pulsed with each corresponding peptide and produced IFN-γ. Theresults for P104 (SEQ ID NO: 4) are shown in FIG. 12A, and the resultsfor P106 (SEQ ID NO: 6) are shown in FIG. 12B. The recognition was notinhibited by anti-CD8 antibody or anti-A24 antibody; however, it wasinhibited weakly by anti-class I antibody, and completely by anti-CD4antibody, anti-A2 antibody, and anti-class II antibody. In addition, apeptide derived from HIV was used as the negative control for thepeptides.

When CD4⁺ cells, which were purified by well-known methods usinganti-CD4 antibodies from the cells that had been stimulated by eachpeptide, were co-cultured with T2 cells that had been pulsed with eachcorresponding peptide, production of IFN-γ was observed (FIG. 13A: P104(SEQ ID NO: 4) and FIG. 13B: P106 (SEQ ID NO: 6)). This IFN-γ productionwas completely inhibited by the anti-class I antibody and by theanti-class II antibody.

It is thus probable that the cells that were induced by the peptidestimulation are not so-called CD8⁺ CTL, but rather CD4⁺ T-lymphocytes.In general, CD4⁺ T-lymphocytes are thought to recognize a complex of anMHC class II molecule and a peptide on an antigen-presenting cell andproduce various cytokines, such as IFN-γ. However, given that productionof IFN-γ was inhibited by the anti-class I antibody, as described above,and based on a recent report of a case in which CD4⁺ cells recognizeantigens in a class I-restricted manner (Cancer Research, 1999, Volume59, pp. 6230-6238), it is possible that the CD4⁺ cells that were inducedin the present example recognize antigens in a class I-restrictedmanner. That is to say, it is suggested that the cells that have beeninduced by each of the peptides are CD4⁺ cells, and that these cellsrecognize each peptide presented on the HLA-A2 molecules that arepresent on the surface of T2 cells and produce IFN-γ. In addition, giventhat the aforementioned production of IFN-γ was inhibited by anti-classII antibodies, the following may be deduced: class II molecules areexpressed on the CD4⁺ cells that have been induced, while the CD4molecule is expressed on T2 cells; the CD4 molecule is known to bind tothe β2 region of an MHC class II molecule, and the binding stabilizesthe T-cell antigen receptor (TCR)/peptide/MHC complex (Cancer Research,1999, Volume 59, pp. 62306238). Accordingly, it can be deduced that thebinding of T2 cells to CD4⁺ cells via a class II molecule is involved inthe enhancement of the stability of the binding of the TCR on CD4⁺ cellsto the complex of the class I molecule on T2 cells and the peptide. Or,there is also the possibility that a peptide with a weak affinity bindsto a class II molecule that is expressed on CD4⁺ cells, and this complexis recognized by a CD4⁺ cell in the vicinity.

The present example was performed based on the results shown in Example6, that is, based on the fact that cells that were induced from the PBMCderived from the MS patient or the healthy subject by each of thepeptides recognized T2 cells that were pulsed with the peptide, but didnot exhibit cytotoxic activity. Therefore, although the PBMC that werederived from healthy subjects were used in the present example, it canbe deduced that in the present example, using PBMC derived from healthysubjects would be the same as PBMC that were derived from MS patients.

Example 8 Detection of Peptide Specific Antibody in Serum of MultipleSclerosis Patients

The presence of immunoglobulin E (IgE) and immunoglobulin G (IgG) thatspecifically recognize P102 (SEQ ID NO: 2), P103 (SEQ ID NO: 3), P104(SEQ ID NO: 4), or P106 (SEQ ID NO: 6) in the serum of a multiplesclerosis patient, a brain tumor patient, and a healthy subject wasmeasured by the enzyme-linked immunosorbent assay (ELISA).

Each aforementioned peptide (20 μg/well) was immobilized in a 96-wellNunc Covalink flat-bottomed plate (Fisher Scientific), usingdisuccinimidyl suberate (PIERCE), following the product instructions.This plate was blocked with Block Ace (Yukijirushi) and washed with0.05% Tween20-PBS; a serum or blood plasma sample was diluted with 0.05%Tween20-Block Ace and added to the plate at 100 μl/well. Afterincubation for 2 hours at 37° C., the plate was washed with 0.05%Tween20-PBS and further incubated for 2 hours at 37° C. with rabbitanti-human IgE antibody (E chain-specific), anti-human IgG antibody (ychain-specific) (DAKO), or anti-human IgG subclass-specific antibody(Zymed Laboratories), each of which were diluted to 1:1000. This platewas washed 9 times; 100 μl of horseradish peroxidase dextran polymerlinked to goat anti-rabbit Ig antibody (EnVision, DAKO) diluted to1:100, was added to each well and further incubated for 40 minutes atroom temperature. After washing, 100 μl of tetramethylbenzene substratesolution (KPL) was added. Thereafter, the reaction was stopped by adding1 M phosphoric acid, and the optical density (OD) at 450 nm wasmeasured. In so doing, peptides derived from EBV, HIV, SART2(JP-11-318455), or SART3 (Cancer Research, 1999, Volume 59, pp.4956-4063) were used as controls and measured similarly. In addition,the OD value arising from a non-specific response obtained by using apeptide derived from HIV was subtracted from the aforementioned ODvalues. Furthermore, when the soluble peptide corresponding to therespective immobilized peptide was added to each antibody measurementsystem, the response was inhibited and the optical density decreased,but no inhibition was observed if an unrelated peptide was added.Therefore, it is revealed that the aforementioned IgE and IgG that weremeasured are specific antibodies against the respective peptides.

The results with respect to the sera of MS patients, brain tumorpatients, and healthy subjects are shown in FIG. 14, FIG. 15, and FIG.16, respectively. In each of the figures, FIG. A shows IgE and FIG. Bshows IgG. FIG. 14A clearly reveals that IgE that specificallyrecognizes P102 (SEQ ID NO: 2) or P104 (SEQ ID NO: 4) is present in theserum of MS patients. However, IgG was not detected. Meanwhile, both IgEand IgG were not detected in the sera of brain tumor patients or healthysubjects.

So far, no report exists in the literature that suggests the involvementof antibodies in MS. This may be due to MS being predominantly apathology of helper T1 cells. Meanwhile, the results from animalexperiments that have been performed based on the analysis of suchpathological mechanisms are in no way linked to the results fromclinical experiments (Martin, R. et al., Nature Immunology, 2001, Volume2, pp. 785-788). Therefore, in terms of MS, a mechanism that has not yetbeen elucidated may be involved in the pathology. In the presentexample, it has been shown that an IgE that recognizes a peptidepertaining to the present invention is present in the blood of the MSpatient. Therefore, it is also possible that the stimulation of mastcells by the IgE that recognizes the aforementioned peptide is relatedto the pathological formation of MS. From the foregoing, preventionand/or treatment of MS is thought to be possible by using the peptidedescribed above, for instance, by methods that induce immunologicaltolerance or the like.

INDUSTRIAL APPLICABILITY

A gene coding for a tumor antigen that is recognized byHLA-A2-restricted tumor-specific cytotoxic T-lymphocytes was isolatedfrom the cDNA library of the human glioma cell strain KNS60, using agene expression cloning method, and identified. Furthermore, a peptideand a polypeptide having the epitope of the tumor antigen were foundbased on the tumor antigen coded by the gene that was obtained.

These peptide and polypeptide induced HLA-A2-restricted tumor-specificcytotoxic T-lymphocytes from peripheral blood mononuclear cells derivedfrom a brain tumor patient, which recognize the peptide and thepolypeptide and may damage tumor cells. The HLA-A2 allele is found inapproximately 40% of Japanese, approximately 53% of Chinese,approximately 49% of North American Caucasians, approximately 38% ofSouth American Caucasians, and approximately 23% of African Blacks.Therefore, according to the present invention, specific immunotherapy ofcancers, for example, brain tumors and the like, is possible, and aconsiderable contribution to cancer therapy can be expected. Inaddition, the present invention also contributes considerably tofundamental research into molecules involved in recognition of cancer bycytotoxic T-lymphocytes.

Furthermore, it was discovered that the peripheral blood mononuclearcells, derived from the multiple sclerosis patient, which have beenstimulated with the peptides of the present invention, recognize eachcorresponding peptide and enhance production of IFN-γ, but do notrecognize the tumor cells; that the production of IFN-γ is due toCD4-positive cells; and that IgE that recognizes the peptide is presentin the serum of multiple sclerosis patients. It is possible that thepeptides of the present invention are involved in multiple sclerosis andmay be utilized in the elucidation, as well as prevention and/ortreatment, of multiple sclerosis.

Sequence Listing Free Text

-   Sequence Listing SEQ ID NO: 1: designed peptide recognized by    HLA-A2-restricted cytotoxic T-lymphocytes.-   Sequence Listing SEQ ID NO: 2: designed peptide recognized by    HLA-A2-restricted cytotoxic T-lymphocytes.-   Sequence Listing SEQ ID NO: 3: designed peptide recognized by    HLA-A2-restricted cytotoxic T-lymphocytes.-   Sequence Listing SEQ ID NO: 4: designed peptide recognized by    HLA-A2-restricted cytotoxic T-lymphocytes.-   Sequence Listing SEQ ID NO: 5: designed peptide recognized by    HLA-A2-restricted cytotoxic T-lymphocytes.-   Sequence Listing SEQ ID NO: 6: designed peptide recognized by    HLA-A2-restricted cytotoxic T-lymphocytes.-   Sequence Listing SEQ ID NO: 7: designed peptide recognized by    HLA-A2-restricted cytotoxic T-lymphocytes.-   Sequence Listing SEQ ID NO: 8: designed peptide recognized by    HLA-A2-restricted cytotoxic T-lymphocytes.-   Sequence Listing SEQ ID NO: 9: designed peptide recognized by    HLA-A2-restricted cytotoxic T-lymphocytes.-   Sequence Listing SEQ ID NO: 10: designed peptide recognized by    HLA-A2-restricted cytotoxic T-lymphocytes.-   Sequence Listing SEQ ID NO: 11: designed peptide recognized by    HLA-A2-restricted cytotoxic T-lymphocytes.-   Sequence Listing SEQ ID NO: 12: designed peptide recognized by    HLA-A2-restricted cytotoxic T-lymphocytes.-   Sequence Listing SEQ ID NO: 13: designed peptide recognized by    HLA-A2-restricted cytotoxic T-lymphocytes.

1. An isolated peptide having the amino acid sequence set forth in anyone of SEQ ID NO: 1 to 13 in the Sequence Listing.
 2. An isolatedpolypeptide having the amino acid sequence set forth in any one of SEQID NO: 14 to 18 in the Sequence Listing. 3-39 (canceled)
 40. An antibodythat immunologically recognizes the polypeptide of claim
 2. 41. A methodfor quantitatively and/or qualitatively measuring: the polypeptide ofclaim
 2. 42. The peptide of claim 1, wherein said peptide is recognizedby a cytotoxic T-lymphocyte and/or induces a cytotoxic T-lymphocyte. 43.The peptide of claim 42, wherein being recognized by a cytotoxicT-lymphocyte and/or inducing a cytotoxic T-lymphocyte is beingrecognized by a cytotoxic T-lymphocyte in an HLA-A2-restricted mannerand/or inducing a cytotoxic T-lymphocyte in an HLA-A2-restricted manner.44. The polypeptide of claim 2, wherein said polypeptide is recognizedby a cytotoxic T-lymphocyte and/or induces a cytotoxic T-lymphocyte. 45.The polypeptide of claim 44, wherein being recognized by a cytotoxicT-lymphocyte and/or inducing a cytotoxic T-lymphocyte, is beingrecognized by a cytotoxic T-lymphocyte in an HLA-A2-restricted mannerand/or inducing a cytotoxic T-lymphocyte in an HLA-A2-restricted manner.46. A medicament comprising at least one peptide of claim
 1. 47. Amedicament comprising at least one polypeptide of claim
 2. 48. A methodto treat cancer comprising administering at least one peptide of claim 1to a patient in need of such treatment.
 49. A method to treat cancercomprising administering at least one polypeptide of claim 2 to apatient in need of such treatment.
 50. A method to treat cancercomprising treating peripheral blood mononuclear cells which haveisolated from said patient, with at least one peptide of claim 1; andadministering the thus treated peripheral blood mononuclear cells tosaid patient.
 51. A method to treat cancer comprising administering themedicament of claim
 46. 52. A method to treat cancer comprisingadministering the medicament of claim
 47. 53. The method according toclaim 48, wherein said cancer is a brain tumor.
 54. A cancer vaccinethat contains an immunoprotective effective amount of at least onepeptide of claim
 1. 55. A cancer vaccine that contains animmunoprotective effective amount of at least one polypeptide of claim2.
 56. A method for inducing a cytotoxic T-lymphocyte comprisingutilizing at least one peptide of claim
 1. 57. A method for inducing acytotoxic T-lymphocyte comprising utilizing at least one polypeptide ofclaim
 2. 58. A method for inducing a cytotoxic T-lymphocyte comprisingcontacting peripheral blood mononuclear cells with at least one peptideof claim
 1. 59. A method for inducing a cytotoxic T-lymphocytecomprising: i) incubating an antigen-presenting cell that retains HLA-A2with a peptide having the amino acid sequence set forth in any one ofSEQ ID NO: 1 to 13 in the Sequence Listing; or ii) expressing apolypeptide having the amino acid sequence set forth in any one of SEQID NO: 14 to 18 in the Sequence Listing in an antigen-presenting cellthat retains HLA-A2; and iii) utilizing the cell obtained in said i) orsaid ii) for stimulating a group of cells that contain a precursor cellof the cytotoxic T-lymphocyte.
 60. A method of treating or preventingmultiple sclerosis comprising administering at least one peptide ofclaim 1 to a patient in need of such treatment.
 61. A method of treatingor preventing multiple sclerosis comprising administering at least onepolypeptide of claim 2 to a patient in need of such treatment.
 62. Amethod of treating or preventing multiple sclerosis comprisingadministering the medicament of claim
 46. 63. A method of treating orpreventing multiple sclerosis comprising administering the medicament ofclaim
 47. 64. An isolated polynucleotide having a nucleotide sequencethat codes for a peptide having the amino acid sequence set forth in anyone of SEQ ID NO: 1 to 13 in the Sequence Listing or a polypeptidehaving the amino acid sequence set forth in any one of SEQ ID NO: 14 to18 in the Sequence Listing, or a complementary nucleotide sequencethereof.
 65. An isolated polynucleotide having the nucleotide sequenceset forth in any one of SEQ ID NO: 19 to 23 in the Sequence Listing, ora complementary nucleotide sequence thereof.
 66. The polynucleotide ofclaim 65, wherein said nucleotide sequence is such that the polypeptidecoded by said nucleotide sequence is recognized by a cytotoxicT-lymphocyte, and/or induces a cytotoxic T-lymphocyte, or acomplementary nucleotide sequence thereof.
 67. The polynucleotide ofclaim 66, wherein being recognized by a cytotoxic T-lymphocyte and/orinducing a cytotoxic T-lymphocyte is being recognized by a cytotoxicT-lymphocyte in an HLA-A2-restricted manner and/or inducing a cytotoxicT cell in an HLA-A2-restricted manner.
 68. An isolated polynucleotidethat hybridizes under stringent conditions with the polynucleotide ofclaim
 64. 69. A recombinant vector comprising the polynucleotideaccording to claim
 64. 70. The recombinant vector of claim 69, whereinsaid recombinant vector is a recombinant expression vector.
 71. Atransformant that has been transformed by the recombinant vector ofclaim
 69. 72. A transformant that has been transformed by therecombinant vector of claim
 70. 73. A method for preparing a peptide ora polypeptide comprising culturing a transformant that has beentransformed by a recombinant vector of claim
 72. 74. An antibody thatimmunologically recognizes the peptide of claim
 1. 75. A method foridentifying a compound that enhances recognition of the peptide of claim43 at least by an HLA-A2-restricted cytotoxic T-lymphocyte, comprisingcontacting HLA-A2⁺ cells which have been pulsed with said peptide, withsaid cytotoxic T lymphocyte which recognize a complex of the peptide andHLA-A2 molecule in the presence or absence of a compound; anddetermining whether said compound enhances said recognition by measuringIFN-γ production from said cytotoxic T lymphocyte.
 76. A method foridentifying a compound that enhances recognition of the peptide of claim43 at least by an HLA-A2-restricted cytotoxic T-lymphocyte, comprisingcontacting HLA-A2⁺ cells which have been transfected with apolynucleotide, with said cytotoxic T lymphocyte which recognize acomplex of the peptide and HLA-A2 molecule in the presence or absence ofa compound; and determining whether said compound enhances saidrecognition by measuring IFN-γ production from said cytotoxic Tlymphocyte, wherein said polynucleotide is an isolated polynucleotidehaving a nucleotide sequence that codes for a peptide having the aminoacid sequence set forth in any one of SEQ ID NO: 1 to 13 in the SequenceListing or a polypeptide having the amino acid sequence set forth in anyone of SEQ ID NO: 14 to 18 in the Sequence Listing, or a complementarynucleotide sequence thereof.
 77. A compound that is identified by themethod of claim
 75. 78. A compound that is identified by the method ofclaim
 76. 79. A compound that enhances the recognition of at least onepeptide of claim 43 by an HLA-A2-restricted cytotoxic T-lymphocyte. 80.A compound that interacts with the polynucleotide according to claim 64and enhances the expression thereof.
 81. A pharmaceutical compositionfor use in cancer therapy that contains an effective dose of at leastone of: the peptide of claim 43, an isolated polypeptide having theamino acid sequence set forth in any one of SEQ ID NO: 14 to 18 in theSequence Listing, an isolated polynucleotide having a nucleotidesequence that codes for a peptide having the amino acid sequence setforth in any one of SEQ ID NO: 1 to 13 in the Sequence Listing or apolypeptide having the amino acid sequence set forth in any one of SEQID NO: 14 to 18 in the Sequence Listing, or a complementary nucleotidesequence thereof, a recombinant vector or recombinant expression vectorcontaining said polynucleotide or the complementary strand thereof, atransformant containing said recombinant vector or recombinantexpression vector, an antibody that immunologically recognizes saidpeptide or polypeptide, a compound that enhances the recognition of atleast one of said peptide or polypeptide by an HLA-A2-restrictedcytotoxic T-lymphocyte, and a compound that interacts with saidpolynucleotide and enhances the expression thereof.
 82. A pharmaceuticalcomposition for use in the prevention and/or treatment of multiplesclerosis that contains an effective dose of at least one of: thepeptide of claim 43, an isolated polypeptide having the amino acidsequence set forth in any one of SEQ ID NO: 14 to 18 in the SequenceListing, the isolated polynucleotide having a nucleotide sequence thatcodes for a peptide having the amino acid sequence set forth in any oneof SEQ ID NO: 1 to 13 in the Sequence Listing or a polypeptide havingthe amino acid sequence set forth in any one of SEQ ID NO: 14 to 18 inthe Sequence Listing, or a complementary nucleotide sequence thereof, arecombinant vector or recombinant expression vector containing saidpolynucleotide or the complementary strand thereof, a transformantcontaining said recombinant vector or recombinant expression vector, anantibody that immunologically recognizes said peptide or polypeptide, acompound that enhances the recognition of at least one of said peptideor polypeptide by an HLA-A2-restricted cytotoxic T-lymphocyte, and acompound that interacts with said polynucleotide and enhances theexpression thereof.
 83. A method for quantitatively and/or qualitativelymeasuring: the peptide of claim
 1. 84. The method of claim 83, which isused in cancer screening.
 85. A reagent kit comprising at least one of:the peptide of claim 43, an isolated polypeptide having the amino acidsequence set forth in any one of SEQ ID NO: 14 to 18 in the SequenceListing, an antibody that immunologically recognizes said peptide orpolypeptide, an isolated polynucleotide having a nucleotide sequencethat codes for a peptide having the amino acid sequence set forth in anyone of SEQ ID NO: 1 to 13 in the Sequence Listing or a polypeptidehaving the amino acid sequence set forth in any one of SEQ ID NO: 14 to18 in the Sequence Listing, or a complementary nucleotide sequencethereof, or a polynucleotide that hybridizes to said polynucleotide orthe complementary strand thereof under stringent conditions, arecombinant vector or recombinant expression vector comprising saidpolynucleotide or the complementary strand thereof, a transformantcomprising said recombinant vector or recombinant expression vector; anda buffered solution.
 86. The reagent kit of claim 85, wherein thereagent kit is used in a method for identifying a compound that enhancesrecognition of said peptide or said polypeptide at least by anHLA-A2-restricted cytotoxic T-lymphocyte.
 87. The reagent kit of claim85, wherein the reagent kit is used in a method for measuringquantitatively and/or qualitatively said peptide, or said polypeptide,or said polynucleotide.
 88. The reagent kit of claim 85, wherein thereagent kit is used in a cancer screening.
 89. A cytotoxic T-lymphocytethat is induced by the method of claim
 56. 90. A cytotoxic T-lymphocyte,which is derived from a peripheral blood mononuclear cell obtained froma brain tumor patient or a multiple sclerosis patient, wherein thecytotoxic T-lymphocyte is induced by the method of claim
 56. 91. Themethod according to claim 49, wherein said cancer is a brain tumor. 92.A compound that enhances the recognition of at least one polypeptide ofclaim 45 by an HLA-A2-restricted cytotoxic T-lymphocyte.
 93. A methodfor quantitatively and/or qualitatively measuring: the polynucleotideaccording to claim
 64. 94. A cytotoxic T-lymphocyte that is induced bythe method of claim
 57. 95. A cytotoxic T-lymphocyte, which is derivedfrom a peripheral blood mononuclear cell obtained from a brain tumorpatient or a multiple sclerosis patient, wherein the cytotoxicT-lymphocyte is induced by the method of claim 57.